Therapeutic compounds containing xanthinyl

ABSTRACT

Therapeutic compounds with at least one carboxylic acid, ester or amide-substituted side chain have the formula: 
     
         CORE MOIETY --(R).sub.j 
    
     wherein j is an integer from one to three. The core moiety is non-cyclic or cyclic (carbocyclic or heterocyclic). R may be selected from among hydrogen, halogen, hydroxyl, amino, substituted or unsubstituted C.sub.(1-10) alkyl, C.sub.(2-10) alkenyl, carbocyclic or heterocyclic groups and at least one R has the formula I: ##STR1## wherein: one or two p are the integer one, otherwise p is two; and n is an integer from three to twenty; R 1  is selected from the group consisting of substituted and unsubstituted CH 2  ; NR 3 , R 3  being hydrogen, substituted or unsubstituted C.sub.(1-20) alkyl, C.sub.(1-20) alkoxyl, C.sub.(2-20) alkenyl or C.sub.(1-20) hydroxyalkyl, or carbocyclic or heterocyclic group; O; --CHR 4  O--, R 4  being substituted or unsubstituted C.sub.(1-20) alkyl, C.sub.(1-20) alkoxyl, C.sub.(2-20) alkenyl, C.sub.(1-20) hydroxyalkyl, or R 2  and R 4  join to form a substituted or unsubstituted heterocycle having four to seven ring atoms, the ether group --O-- of --CHR 4  O-- being a member of the heterocycle. R 2  is selected from the group consisting of hydrogen; halogen; substituted or unsubstituted C.sub.(1-10) alkyl; C.sub.(1-10) alkoxyl; C.sub.(2-10) alkenyl; C.sub.(1-10) hydroxyallyl; --A(R 5 ) m , A being N or O, m being one or two and R 5  being hydrogen, a substituted or unsubstituted C.sub.(1-10) alkyl, C.sub.(1-10) alkoxyl, C.sub.(2-10) alkenyl or C.sub.(1-10) hydroxyalkyl), or carbocyclic or heterocyclic group. At least one of R 1  is NR 3 , O or --CHR 4  O--, or R 2  is --A(R 5 ) m . The compounds and pharmaceutical compositions thereof are useful as therapies for diseases advanced via intracellular signaling through specific intracellular signaling pathways by mediating a signaling response to an external stimuli.

CROSS REFERENCE TO RELATED APPLICATION

This is a Continuation-in-Part patent application of U.S. patentapplication Ser. No. 08/199,368, which was filed on Feb. 18, 1994 andwhich is now abandoned.

TECHNICAL FIELD OF THE INVENTION

The invention provides a group of compounds that are effective agentsfor inhibiting specific cellular signaling events often induced byinflammatory stimuli, or to be directly or indirectly antimicrobial toyeast or fungal infections. More specifically, the inventive compoundshave at least one carboxylic acid, ester or amide-substituted chainbonded to a core moiety. The inventive compounds are, among otherthings, useful antagonists to control intracellular levels of specificsn-2 unsaturated phosphatidic acids and corresponding phosphatidicacid-derived diacylglycerols, intracellular cell signaling messengerswhich occur in response to pro-inflammatory proliferative stimuli.

BACKGROUND OF THE INVENTION

Pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine), abbreviated PTX,is a xanthine derivative which has seen widespread medical use for theincrease of blood flow. PTX is disclosed in U.S. Pat. Nos. 3,422,107 and3,737,433, both to Mohler et al. Metabolites of PTX were summarized inDavis et al., Applied Environment Microbiol. 48:327, 1984. A metaboliteof PTX is 1-(5-hydroxyhexyl)-3,7-dimethylxanthine, designated M1. M1-wasalso disclosed as increasing cerebral blood flow in U.S. Pat. Nos.4,515,795 and 4,576,947 to Hinze et al. Other metabolites,1-(5-pentyl)-3,7-dimethylxanthine carboxylic acid, designated M5, and1-(4-butyl)-3,7-dimethylxanthine carboxylic acid, designated M5, weredisclosed by Bryce et al., Arzneim.-Forsch./Drug Res. 39(4):512-517,1989. In addition, U.S. Pat. Nos. 4,833,146 and 5,039,666 to Gebert etal. and Novick, Jr., respectively, disclose use of tertiary alcoholanalogs of xanthine for enhancing cerebral blood flow.

PTX and its known metabolites thereof have been shown to have in vivoactivity in specific biologic systems. U.S. Pat. No. 4,636,507 toKreutzer et al. describes an ability of PTX and M1, to further promotechemotaxis in polymorphonuclear leukocytes responding to a chemotaxisstimulator. In addition, PTX and related tertiary alcohol substitutedxanthines inhibit activity of certain cytokines to affect chemotaxis(U.S. Pat. Nos. 4,965,271 and 5,096,906 to Mandell et al.). Byadministrating PTX and GM-CSF, patients undergoing allogeneic bonemarrow transplant exhibited decreased levels of tumor necrosis factor,TNF, (Bianco et al., Blood 76: Supplement 1 (522A), 1990). Reduction inassayable levels of TNF was accompanied by a reduction in bone marrowtransplant-related complications. However, in normal volunteers, TNFlevels were higher among PTX recipients. Therefore, elevated levels ofTNF are not the primary cause of such complications.

Further research with PTX, its metabolites and their activity relatingto various biologic systems spurred investigations with potentialtherapeutic agents heretofore unknown. These agents were identified aspotential therapies for treating or preventing disease by inhibitingsecondary cellular response to an external or in situ primary stimuli.These investigations sought to identify efficacious therapeuticcompounds which were safe and effective for human or animaladministration and maintain cellular homeostasis in the face of avariety of inflammatory stimuli.

In undertaking these investigations, previously unknown therapeuticcompounds were discovered. These novel compounds are discussed herein.These compounds exhibit remarkable characteristics in predictive invitro disease assays, which known compounds do not possess, indicatingefficacious therapies for treating or preventing disease using theinventive compounds.

SUMMARY OF THE INVENTION

The invention provides carboxylic acid, ester and amide-substitutedtherapeutic compounds and pharmaceutical compositions and uses thereof.The inventive carboxylic acid, ester or amide-substituted compounds areuseful in a large variety of therapeutic indications for treating orpreventing disease. In particular, the inventive compounds andpharmaceutical compositions thereof provide therapy for diseases causedor advanced by intracellular signaling through specific intracellularsignaling pathways, specifically the pathways discussed herein, bymediating a signaling response to an external stimuli.Abnormally-induced intracellular signaling is characteristic of diseasestreatable using the inventive compounds or pharmaceutical compositionsthereof.

The inventive compounds have at least one carboxylic acid, ester oramide-containing side chain and are preferably carbocyclic orheterocyclic compounds. The inventive compounds and pharmaceuticalcompositions thereof have the formula:

    CORE MOIETY --(R).sub.j

including resolved enantiomers and/or diastereomers, hydrates, salts,solvates and mixtures thereof, wherein j is an integer from one tothree, the core moiety is non-cyclic or cyclic (carbocyclic orheterocyclic) and R may be selected from among: hydrogen, halogen(preferably bromine, chlorine, fluorine and iodine), hydroxyl, amino,substituted or unsubstituted C.sub.(1-10) alkyl, C.sub.(2-10) alkenyl,carbocyclic or heterocyclic groups and formula I. The inventivecompounds have at least one R of the following formula I: ##STR2##wherein: one or two p are the integer one, otherwise p is two;

n is an integer from three to twenty, preferably seven to sixteen, mostpreferably five to sixteen.

R₁ can be selected from the group consisting of substituted andunsubstituted CH₂ ; NR₃, R₃ being hydrogen, substituted or unsubstitutedC.sub.(1-20) alkyl, C.sub.(1-20) alkoxyl, C.sub.(2-20) alkenyl orC.sub.(1-20) hydroxyalkyl, or carbocyclic or heterocyclic group; O;--CHR₄ O--, or --C(R₄)_(r) O--, r being one or two, R₄ being ═O,hydrogen, substituted or unsubstituted C.sub.(1-20) alkyl, C.sub.(1-20)alkoxyl, C.sub.(2-20) alkenyl, C.sub.(1-20) hydroxyalkyl, C.sub.(1-20)aminoalkyl, --(CH₂)_(q) A(R₅)_(m), q being an integer from one to four,A being N or O, m being one or two and R₅ being hydrogen, a substitutedor unsubstituted C.sub.(1-10) alkyl, C.sub.(1-10) alkoxyl, C.sub.(2-10)alkenyl or C.sub.(1-10) hydroxyalkyl, C.sub.(1-10) aminoalkyl,carbocyclic or heterocyclic group, or R₂ and R₄ join to form asubstituted or unsubstituted heterocycle having four to seven ringatoms, the --O-- of --CHR₄ O-- being a member of the heterocycle.

R₂ can be selected from the group consisting of hydrogen; halogen;substituted or unsubstituted C.sub.(1-10) alkyl; C.sub.(1-10) alkoxyl;C.sub.(2-10) alkenyl; C.sub.(1-10) hydroxyalkyl, C.sub.(1-20)aminoalkyl; --A(R₅)_(m) ; --CHR₆ A(R₅)_(m) ; A, R₅ and m being definedabove, R₆ being a substituted or unsubstituted C.sub.(1-20) alkyl,C.sub.(1-20) alkoxyl, C.sub.(2-20) alkenyl, C.sub.(1-20) hydroxyalkyl,C.sub.(1-20) aminoalkyl, carbocyclic or heterocyclic groups, or A is N,m is two and the two R₅ join to form a substituted or unsubstitutedheterocycle having from four to seven ring atoms, A comprising a heteroatom of the heterocycle.

In the inventive compounds, at least one of R₁ is NR₃, O or --CHR₄ O--,or R₂ is --A(R₅)_(m). Optionally, (CH₂)_(n) may 1) be substituted by ahalogen, hydroxide, substituted or unsubstituted C.sub.(1-10) alkyl,C.sub.(2-10) alkenyl, C.sub.(1-10) alkoxyl, C.sub.(1-10) acyloxy,C.sub.(1-10) oxyalkyl, carbocyclic or heterocyclic group; 2) have one ortwo unsaturated bonds (preferably in a cis configuration); or 3) beinterrupted by at least one oxygen atom.

A non-cyclic core moiety may include, but is not limited to, forexample, acetamide, amide, amine, amino acid (one or two), carboxide,ester, terminal halogen or hydrogen atom, hydroxide, glutaric acid,glycine derivative, ketone, phosphate, phosphonate, sulfate, sulfonate,sulfone, sulfoxide, simple ionic functional group, thiol, thiolester orthe like. Exemplary core moiety amino acids may include, but are notlimited to, one or more of the following: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine. The non-cyclic core moietymay preferably be an amide, carboxyl ester, carboxide, hydrogen,hydroxide or a dipeptide comprising two amino acids selected from theforegoing exemplary list. A non-cyclic, halogen-core moiety may be, forexample, bromine, chlorine, fluorine or iodine.

A cyclic core may be at least one five- to seven-member,non-heterocyclic ring (i.e., carbocycle) or a heterocycle. The at leastone five- to seven-membered cyclic core may preferably have from one tothree, five- to six-membered ring structures in a predominantly planarconfiguration. An exemplary, non-heterocyclic ring core moiety may beselected from the group consisting of substituted or unsubstitutedbenzene; biphenyl; cyclohexane; cyclohexanedione; cyclopentanedione;napthlalene; phenol; quinone; salicylic acid; stilbene andtricyclododecane.

Although other heterocyclic cores are within the scope of the invention,the following representatives are preferred: substituted orunsubstituted barbituric acid; benzamide; lactam; glutarimide;homophthalimide; hydrophthalimide; imidazole; imidazole amide;indomethacin; isocarbostyril; lumazine; N-alkylheterocyclic;N-heterocyclic; pteridine; pthalimide; piperidine; pyridine; pyrimidine;pyrrole amide; quaternized N-heterocyclic; quinolizinedione;quinazolinone; quinoline; recorsinol; succinimide; theobromine; thymine;triazine; uric acid; uracil; vitamins A, E or K; or xanthine.

Preferably, R is bonded to a nitrogen of the core moiety, if present,most preferably to the nitrogen of a glutarimide, methylthymine,thymine, uracil or xanthine core. In representative, preferredcompounds, R having formula I may be bonded to an N₁ nitrogen ofxanthine (and N₃ and N₇ xanthine nitrogens may be independentlysubstituted by a member selected from the group consisting of hydrogen,C.sub.(1-6) alkyl, fluoro, chloro and amino); or N₁ nitrogen of uracil.Alternatively, R having formula I may be bonded to N₁ and N₃ xanthinenitrogens and the N₇ xanthine nitrogen is substituted by a memberselected from the group consisting of hydrogen, methyl, fluoro, chloroand amino. Representative, preferred inventive compounds are compoundsof formulas II, III and IV: ##STR3## wherein R is as defined above.

The invention also provides a pharmaceutical composition. Pharmaceuticalcompositions of the inventive compounds comprise a pharmaceuticalcarrier or diluent and some amount of an inventive compound. The natureof the composition and the pharmaceutical carrier or diluent will, ofcourse, depend upon the intended route of administration, for example,parenterally, topically, orally or by inhalation for treatment of apatient with disease symptoms.

The invention also provides a method for treating an individual having avariety of diseases. The disease is characterized by or can be treatedby inhibiting an immune response or a cellular response to external orin situ primary stimuli. Treatment of the disease states involvesmediating the cellular response through a specific phospholipid-basedsecond messenger acting adjacent to a cell membrane inner leaflet. Thesecond messenger pathway is activated in response to various noxious orproliferative stimuli, characteristic of disease states treatable usingthe inventive compounds or pharmaceutical compositions thereof.Biochemistry of this second messenger pathway is described herein. Morespecifically, the invention includes methods for treating or preventingclinical symptoms of various disease states or reducing toxicity ofother treatments by inhibiting cellular signaling through a secondmessenger pathway involving signaling through phosphatidic acid andthrough glycan phosphatidylinositol (Gly PI).

Gly PI consists of a phosphatidylinositol-1-phosphate (PIP) boundthrough the carbon 6-hydroxyl to a glucosamine residue, which in turn isbound, usually to 2-5 other glycan residues (1→4 type, linear bonds)containing an additional one to three phosphoethanolamine moieties, thelast of which may be bound to an external protein such as Thy-1.Evidence suggests a broad variety of structural variation in the sn-1and sn-2 positions of the glycerol/lipid moiety of thephosphatidylinositol, as well as fatty acyl addition to the 2-OH groupof the inositol. Several functional parameters of structure have beenobserved, the most remarkable of which point to a minimum presence of atleast one myristoyl sidechain in Gly-PI molecules, the presence of bothalkyl (ether) and acyl chains in the sn-l position, and the presence ofpalmitate (C16:0) in the 2-OH position of the inositol inprotein-binding Gly-PI. Thomas et al., Biochemistry 29: 5413-5422(1991).

Recent research has demonstrated that 2-OH-acylation of the inositolmoiety conveys resistance to hydrolysis with Gly PI-directedphospholipase C (P_(i) G-PLC, a phosphodiesterase which hydrolyzes GlyPI to glycan inositol phosphate and diacylglycerol) but not to GlyPI-directed phospholipase D (P_(i) G-PLD, a phosphodiesterase whichhydrolyzes Gly PI to glycan inositol+phosphatidic acid).

Research has identified two functions of Gly-PI: 1) external proteinbinding, the purpose of which may be simple binding to the cell membraneor placement of conformational constraints on the structure ofexternally bound membrane proteins (e.g., so that a particular portionof the molecule faces an extracellular environment); and 2) signaltransduction, including part of the intracellular signal sent by insulinand a detectable portion of the signal transduced by Interleukin-2(IL-2). We have found that signal transducing Gly-PI in B lymphocytes ishydrolyzed following anti-mu crosslinking, and then resynthesizedrapidly. In these systems, two Gly-PI species are synthesized: a)GlyPI₁, containing 1-myristoyl 2-palmitoyl, 1-o-tetradecanyl (myristyl)2-palmitoyl and 1-myristyl 2-myristyl phosphatidylinositol; and b) GlyPI₂, containing 1-myristoyl 2-oleoyl and 1-o-myristyl 2-linoleoylphosphatidylinositol. Fraction (a) above contains a 1:1 mole content ofC22 or C20 acyl groups attached to the inositol phosphate. The Gly-PI₁fraction, identified by glucosamine labeling followed by massspectrometry, exhibits a characteristic tripartite peak(glycan-inositol: 2-OH-acyl: phosphatidic acid moieties) and isuniformly inositol 2-OH acylated. Therefore, fraction (a) conveysresistance to P_(i) G-PLC but not to P_(i) G-PLD, suggesting that theobserved fraction, when hydrolyzed, will generate 1-myristyl and1-o-myristyl phosphatidic acid species, subsequently observed.

Thus, inventive compounds, useful in treating diseases and reducingtoxicity of other disease treatments, would affect cellular signalingthrough a second messenger pathway by interacting with binding and/orsignaling functions of Gly PI.

A disease state or treatment-induced toxicity are selected from thegroup consisting of: tumor progression involving tumor stimulation ofblood supply (angiogenesis) by production of fibroblast growth factor(FGF), vascular endothelial growth factor (VEGF) or platelet-derivedgrowth factor (PDGF); tumor invasion and formation of metastases throughadhesion molecule binding, expressed by vascular endothelial cells (VCAMand ICAM); tissue invasion through tumor metalloprotease production suchas MMP-9; autoimmune diseases caused by dysregulation of the T cell or Bcell immune systems, treatable by suppression of the T cell or B cellresponses; acute allergic reactions including, but not limited to,asthma and chronic inflammatory diseases, mediated by pro-inflammatorycytokines including tumor necrosis factor (TNF) and IL-1, and rheumatoidarthritis, osteoarthritis, multiple sclerosis or insulin dependentdiabetes mellitus (IDDM), associated with enhanced localization ofinflammatory cells and release of inflammatory cytokines andmetalloproteases; smooth muscle cell, endothelial cell, fibroblast andother cell type proliferation in response to growth factors, such asPDGF-AA, BB, FGF, EGF, etc. (i.e., atherosclerosis, restenosis, stroke,and coronary artery disease); activation of human immunodeficiency virusinfection (AIDS and AIDS related complex); HIV-associated dementia;kidney mesangial cell proliferation in response to IL-1, MIP-1α, PDGF orFGF; inflammation; kidney glomerular or tubular toxicity in response tocyclosporin A or amphotericin B treatment; organ toxicity (e.g.,gastrointestinal or pulmonary epithelial) in response to a cytotoxictherapy (e.g., cytotoxic drug or radiation); effects of non-alkylatinganti-tumor agents; inflammation in response to inflammatory stimuli(e.g., TNF, IL-1 and the like) characterized by production ofmetalloproteases or allergies due to degranulation of mast cells andbasophils in response to IgE or RANTES; bone diseases caused byoverproduction of osteoclast-activating factor (OAF) by osteoclasts; CNSdiseases resulting from over-stimulation by pro-inflammatoryneurotransmitters such as, acetylcholine, serotonin, leuenkephalin orglutamate; acute inflammatory diseases such as septic shock, adultrespiratory distress syndrome; multi-organ dysfunction associated withinflammatory cytokine cascade; and combinations thereof.

In a large number of cells, signaling is dependent upon generation of abroad variety of PA species, some of which are generated from lyso-PA bythe enzyme lyso-PA acyl transferase and some of which are generated from2--O-- acyl glycan-PI by P_(i) G-PLD. Generation of each of these PAspecies (the predominant forms being: 1-acyl and 1-alkyl 2-linoleoyl PAcompounds, generated by LPAAT; and 1-myristyl 2-palmitoyl and1-o-myristyl 2-palmitoyl, generated by P_(i) G-PLD) serves to effectboth proliferative and/or inflammatory signaling in the diseasesdiscussed and cell systems described above.

The inventive compounds are of particular significance for inhibitingIL-2-induced proliferative response. IL-2 signaling inhibition ispotentially useful in the treatment of numerous disease states involvingT-cell activation and hyperproliferation. Exemplary autoimmune diseasestreated by inhibiting IL-2 signaling are lupus, scleroderma, rheumatoidarthritis, multiple sclerosis, glomerula nephritis as well as potentialmalignancies, including but not limited to, chronic myelogenous leukemiaas well as others.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are dose response curves prepared from results in a murinethymocyte assay, determining inhibitive effects of inventive compoundsnos. 3546 and 3549, (see below for chemical name and structure)respectively, on proliferation of thymocytes co-stimulated by ConA andIL-2.

FIGS. 3 and 4 are plotted graphs of compound concentrations (μM) versusinhibition (as a function of incorporated thymidine, cpm) for compoundsnos. 1514 and 1583, respectively, in a mixed lymphocyte reaction (MLR)assay.

FIG. 5 reports the experimentally calculated IC50 values obtained in thean assay investigating inhibitive effects of various inventive compoundson proliferation of Balb/3T3 cells in response to stimulation by PDGF.In addition, FIG. 5 reports LD50 values for each inventive compoundtested in the proliferation assay. The reported LD50 values wereobtained in a corresponding viability assay.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides a genus of compounds which can control cellularbehavior by a particular phase of a secondary messenger pathway system(Bursten et al., J Biol. Chem. 266:20732, 1991). The second messengersare lipids or phospholipids and use the following abbreviations:

PE=phosphatidyl ethanolamine

LPE=lysophosphoethanolamine

PA=phosphatidic acid

LPA=lysophosphatidic acid

DAG=diacylglycerol

LPLD=lysophospholipase-D

LPAAT=lysophosphatidic acid acyl transferase

PAPH=phosphatidic acid phosphohydrolase

PLA2=phospholipase A2.

PLD=phospholipase D

PAA=phosphoarachidonic acid

PC=phosphatidyl choline

"remodeled" PA, cyclic pathway=PAA, LPA, PA and DAG intermediatessubstituted with 1-saturated, 2-linoleoyl or 1,2-dioleoyl,dioleoyl/1,2-sn-dilinoleoyl at the indicated sn-1 and sn-2 positions.

"Classical PI Pathway"=PI, DAG, PA intermediates substituted with1-stearoyl, 2-arachidonoyl fatty acyl side chains.

"PLD-generated PA"=PE, PC, LPA, PA and DAG intermediates substitutedwith, e.g., 1,2-sn-dioleoyl-, 1-alkyl, 2-linoleoyl-, and 1-alkyl,2-docosahexaenoyl-side chains.

Lysophosphatidic acid transferase (LPAAT) effects the synthesis ofphosphatidic acid (PA) from lysophosphatidic acid (LPA) by incorporationof an acyl group from acyl CoA. Hydrolysis of the phosphate moiety by PAphosphohydrolase (PAPH) results in the formation of DAG. These aspectsof the pathway appear to be activated immediately (within a minute) uponstimulation by a primary stimulus (e.g., a cytokine such as IL-1, IL-2or TNF) acting at a receptor on a cellular surface. An immediatedetectable effect is an elevation of levels of PA and DAG.Administration of the compounds of the invention reverse this elevation.

The compounds and pharmaceutical compositions of the invention include,but are not limited to, inhibitors of subspecies of LPAAT and PAPHenzymes with substrate specificity for intermediates with1,2-diunsaturated and 1-alkyl, 2-unsaturated subspecies. Onerepresentative example of such an inhibitor (although not within thegenus of inventive compounds) is PTX. PTX blocks PAPH in a specificactivation pathway that does not involve PI but rather derives from a PAthat is largely composed of 1,2-diunsaturated and 1-alkyl, 2-unsaturatedsubspecies. This was shown, for example, by the demonstration that humanmesangial cells stimulated with TNF produce DAG from PI and regeneratePI in the absence and the presence of PTX. In the latter system there isno evidence to suggest that PA or DAG are derived from sources otherthan PI. It should be emphasized that the compounds of the inventionaffect that subset of PAPH and LPAAT that relates to substrates withunsaturated fatty acids other than arachidonate in the sn-2 position,not the housekeeping forms of these enzymes that serve the PI pathway.

Each membrane phospholipid subclass (e.g., PA, PI, PE, PC and PS)reaches a stable content of characteristic fatty acyl side chains due tocyclic remodeling of the plasma membrane as well as turnover for eachsubclass. PA is often stable, but present in relatively smallquantities. PA in resting cells consists mostly of saturated acylchains, usually consisting of myristate, stearate and palmitate. Inresting cells, PC's acyl side chains consist mostly of acyl palmitate inthe sn-1 position and oleate in the sn-2 position. PE and PI arepredominantly composed of sn-1 stearate and sn-2 arachidonate.

Due to this characteristic content of acyl groups in the sn-1 and sn-2positions, the origin of any PA species-may be deduced from the chemicalnature of its acyl groups in the sn-1 and sn-2 positions. For example,if PA is derived from PC through action of the enzyme PLD, the PA willcontain the characteristic acyl side chains of PC substrate passedthrough the second messenger pathway. Further, the origin of any 1,2sn-substrate species may be differentiated as to its origin. It isimportant to know whether or not each phospholipid species passesthrough a PA form prior to hydrolysis to DAG. The lyso-PA that isconverted to PA and then to DAG may be shown. The complexities of thissecond messenger pathway can be sorted by suitable analyses using fattyacyl side chain chemistry (e.g., by thin layer chromatography,gas-liquid chromatography, or high pressure liquid chromatography) ofintermediates in cells at various time points after stimulation of thesecond messenger pathway.

In certain mesenchymal cells, such as neutrophils and rat or humanmesangial cells, several signaling pathways may be activated in tandem,simultaneously or both. For example, in neutrophils, F-Met-Leu-Phestimulates formation of PA through the action of PLD, followed in timeby formation of DAG through PAPH action. Several minutes later, DAG isgenerated from PI through the classical phosphoinositide pathway. Inmany cells, DAG is derived from both PA that is remodeled through acycle whereby PA is sn-2 hydrolyzed by PLA2, followed by sn-2transacylation by LPAAT and PA that is generated in a PLD-pathway fromeither PE or PC or both substrates by PLD.

The present second messenger pathway involves substrates withunsaturated fatty acids in the sn-2 position other than arachidonate andthose sub-species of PAPH and LPAAT that are not involved in normalcellular housekeeping functions that are part of the classical PIpathway. The PAPH and LPAAT enzymes involved in this specific secondmessenger pathway are exquisitely stereo-specific for different acylside chains and isomeric forms of substrates. Therefore, the inventivecompounds may preferably be substantially enantiomerically pure.

PTX (in vitro) blocks formation of remodeled PA through the PA/DAGpathway at high PTX concentrations (greater than those that could beachieved in patients without dose-limiting side effects) by blockingformation of PA subspecies at LPAAT. Even in the presence of PTX, cellscontinue to form PA through the action of PLD, and DAG is also formedthrough the action of phospholipase C on PC and PI. The latter pathwayare not inhibited by the inventive compounds or PTX. In PTX-treatedcells, DAG derived from remodeled and PLA-generated PA is diminished(e.g., 1,2-sn-dioleoyl DAG, 1-alkyl, 2-linoleoyl DAG and 1-alkyl,2-docosahexaneolyl DAG). Therefore, the inventive compounds and PTXinhibit the formation of only a certain species of PA and DAG byselectively inhibiting a specific second messenger pathway that is onlyactivated in cells by noxious stimuli, but is not used to signal normalcellular housekeeping functions.

Therapeutic Uses of the Inventive Compounds

The specific activation inhibition of the second messenger pathway, asdescribed above and activated primarily by various noxious stimuli,suggests that the inventive compounds are useful in treating a widevariety of clinical indications, mediated at the cellular level by acommon mechanism of action. Moreover, in vitro and in vivo datapresented herein provides predictive data that a wide variety ofclinical indications, having similar effects on the specific secondmessenger pathway (activated by noxious stimuli and mediated through,for example, inflammatory cytokines), may be treated by the inventivecompounds, which specifically inhibit the pathway. In fact, themechanism of action for the inventive compounds explains why thesecompounds have multifarious clinical indications.

Activation of the second messenger pathway is a major mediator ofresponse to noxious stimuli and results in cellular signals that leadto, for example, acute and chronic inflammation, immune response andcancer cell growth. Although the inventive compounds may desirablyinhibit other noxious stimuli not discussed, they most effectivelymediate the above conditions. Signals mediated by the present secondmessenger pathway include, for example, those cellular responses of LPSdirectly; T cell activation by antigen; B cell activation by antigen,cellular responses to IL-1, mediated through the IL-1 Type I receptor(but not the IL-1 Type II receptor), and TNF (Type I receptor), growthstimulated by transformations including, but not limited to, activatedoncogenes (e.g., ras, abl, her 2-neu and the like), smooth muscle cellproliferation stimulated by PDGF, b-FGF and IL-1; T cell and B cellgrowth stimulation by IL-2, IL-4 or IL-7 and IL-4 or IL-6, respectively;and more generally, T cell receptor signaling.

In vitro, the inventive compounds: (1) block IL-1 signal transductionthrough the Type 1 receptor as shown, for example, by preventing IL-1and IL-1 plus PDGF (platelet derived growth factor) induction ofproliferation of smooth muscle, endothelial and kidney mesengial cells;(2) suppress up-regulation of adhesion molecules as shown, for example,by blocking VCAM in endothelial cells; (3) inhibit TNF, LPS and IL-1induced metalloproteases (an inflammation model); (4) block LPS, TNF orIL-1 induced metalloprotease and secondary cytokine production (forprevention and treatment of septic shock); (5) suppress T cell and Bcell activation by antigen, for example, IL-2 and IL-4; (6) inhibit mastcell activation by IgE; (7) are cytotoxic for transformed cells andtumor cell lines, yet not for normal cells; and (8) block signaling byIL-2, IL-4, IL-6 and IL-7 on T and B cells.

The foregoing in vitro effects give rise to the following in vivobiological effects, including, but not limited to: protection andtreatment of endotoxic shock and sepsis induced by gram positive or gramnegative bacteria; inhibition of tumor cell growth; synergisticimmunosuppression, active in autoimmune diseases and in suppressingallograft reactions; and stimulation of hair grow through reversal of anapoptotic process. The inventive compounds are most potent when used toprevent and treat septic shock, treat acute and chronic inflammatorydisease, treat or prevent an autoimmune disease and stimulate hairgrowth (when applied topically).

The inventive compounds also are useful as an adjuvant to inhibit toxicside effects of drugs whose side effects are mediated through thepresent second messenger pathway.

Metalloproteases mediate tissue damage such as glomerular diseases ofthe kidney, joint destruction in arthritis, and lung destruction inemphysema, and play a role in tumor metastases. Three examples ofmetalloproteases include a 92 kD type V gelatinase induced by TNF, IL-1and PDGF plus bFGF, a 72 Kd type IV collagenase that is usuallyconstitutive and induced by TNF or IL-1, and a stromelysin/PUMP-1induced by TNF and IL-1. The inventive compounds can inhibit TNF or IL-1induction of the 92 kD type V gelatinase inducable metalloprotease.Moreover, the inventive compounds can reduce PUMP-1 activity induced by100 U/ml of IL-1. Accordingly, the inventive compounds prevent inductionof certain metalloproteases induced by IL-1 or TNF and are not involvedwith constitutively produced proteases (e.g., 72 kD type IV collagenase)involved in normal tissue remodeling.

The inventive compounds inhibit signal transduction mediated through theType I IL-1 receptor, and are therefore considered as IL-1 antagonists.A recent review article entitled "The Role of Interleukin-1 in Disease"(Dinarello et al., N. Engl. J. Med. 328, 106, Jan. 14, 1993) describedthe role of IL-1 as "an important rapid and direct determinant ofdisease . . . In septic shock, for example, IL-1 acts directly on theblood vessels to induce vasodilatation through the rapid production ofplatelet activating factor and nitric oxide, whereas in autoimmunedisease it acts by stimulating other cells to produce cytokines orenzymes that then act on the target tissue." The article describes agroup of diseases that are mediated by IL-1, including sepsis syndrome,rheumatoid arthritis, inflammatory bowel disease, acute and myelogenousleukemia, insulin-dependent diabetes mellitus, atherosclerosis and otherdiseases including transplant rejection, graft versus host disease(GVHD), psoriasis, asthma, osteoporosis, periodontal disease, autoimmunethyroiditis, alcoholic hepatitis, premature labor secondary to uterineinfection and even sleep disorders. Since the inventive compoundsinhibit cellular signaling through the IL-1 Type I receptor and are IL-1antagonists, the inventive compounds are useful for treating all of theabove-mentioned diseases.

For example, for sepsis syndrome, the mechanism of IL-1-induced shockappears to be the ability of IL-1 to increase the plasma concentrationsof small mediator molecules such as platelet activating factor,prostaglandin and nitric oxide. These substances are potent vasodilatorsand induce shock in laboratory animals. Blocking the action of IL-1prevents the synthesis and release of these mediators. In animals, asingle intravenous injection of IL-1 decreases mean arterial pressure,lowers systemic vascular resistance, and induces leukopenia andthrombocytopenia. In humans, the intravenous administration of IL-1 alsorapidly decreases blood pressure and doses of 300 ng or more perkilogram of body weight may cause severe hypotension. The therapeuticadvantage of blocking the action of IL-1 resides in preventing itsdeleterious biological effects without interfering with the productionof molecules that have a role in homeostasis. The present inventivecompounds address this need, identified by Dinarello et al., byinhibiting cellular signaling only through the IL-1 Type I receptor andnot through the IL-1 Type II receptor.

With regard to rheumatoid arthritis, Dinarello and Wolff state:"Interleukin-1 is present in synovial lining and synovial fluid ofpatients with rheumatoid arthritis, and explants of synovial tissue fromsuch patients produce IL-1 in vitro. Intraarticular injections ofinterleukin-1 induce leukocyte infiltration, cartilage breakdown, andperiarticular bone remodeling in animals. In isolated cartilage and bonecells in vitro, interleukin-1 triggers the expression of genes forcollagenases as well as phospholipases and cyclooxygenase, and blockingits action reduces bacterial-cell-wall-induced arthritis in rats."Therefore, the inventive compounds, as IL-1 antagonists, are useful totreat and prevent rheumatoid arthritis.

With regard to inflammatory bowel disease, ulcerative colitis andCrohn's disease are characterized by infiltrative lesions of the bowelthat contain activated neutrophils and macrophages. IL-1 can stimulateproduction of inflammatory eicosanoids such as prostaglandin E₂ (PGE₂),leukotriene B₄ (LTB₄) and IL-8, an inflammatory cytokine withneutrophil-chemoattractant and neutrophil-stimulating properties. Tissueconcentrations of PGE2 and LTB4 correlate to severity of disease inpatients with ulcerative colitis, patients with inflammatory boweldisease having high tissue concentrations of IL-1 and IL-8. Therefore,an IL-1 antagonist, such as the inventive compounds, would be effectiveto treat inflammatory bowel disease.

With regard to acute and chronic myelogenous leukemia, there isincreasing evidence that IL-1 acts as a growth factor for such tumorcells. Therefore, the inventive compounds should be effective to preventthe growth of worsening of disease for acute and chronic myelogenousleukemias.

Insulin-dependent diabetes mellitus (IDDM) is considered to be anautoimmune disease with destruction of beta cells in the islets ofLangerhans, mediated by immunocompetent cells. Islets of animals withspontaneously occurring IDDM (e.g., BB rats or NOD mice) haveinflammatory cells that contain IL-1. Therefore, the inventive compoundsshould be useful for the preventing and treating IDDM.

IL-1 also plays a role in atherosclerosis development. Endothelial cellsare a target of IL-1. IL-1 stimulates proliferation of vascular smoothmuscle cells. Foam cells, isolated from fatty arterial plaques fromhypercholesterolemic rabbits, contain IL-1β and IL-1β messenger RNA. Theuptake of peripheral blood monocytes results in initiation of IL-1production by these cells. IL-1 also stimulates production of PDGF.Taken together, IL-1 plays a part in the development of atheroscleroticlesions. Therefore, an IL-1 antagonist, such as the inventive compoundsshould be useful in preventing and treating atherosclerosis.

IL-1 activates (through the Type I IL-1 receptor) a lyso-PAacyltransferase (LPAAT) and phosphatidate phosphohydrolase within 5seconds of cell (for example, human mesangial cells, HMC) exposure tothis cytokine. As discussed in detail above, activation of both enzymesresults in production of PA species with sn-1 and sn-2 unsaturated acylgroups, with the majority of sn-2 acyl chains being polyunsaturated.Both IL-1 and a product of LPAAT, 1,2-sn-dilinoleoyl PA, activate asignaling pathway involving hydrolysis of PE to PA. This reaction isfollowed by dephosphorylation of PA to produce both1,2-sn-diacylglycerol, and 1-o-alkyl, or 1-o-alkenyl,acylglycerol (AAG)species. The inventive compounds exert their activity by inhibiting oneor both enzymes at an inner leaflet of the plasma membrane. Therefore,appropriate in vitro models for drug activity may measure inhibition ofstimulation caused by a proinflammatory cytokine or other inflammatorycellular signal.

The generation of the sn-2 unsaturated PA fraction by LPAAT serves toactivate either G-proteins, or acts directly upon PLD through alterationof its lipid microenvironment. Activation of LPAAT and generation of thesn-2-unsaturated PA species is an energy sensitive pathway of PLD. Thisprovides a mechanism for a limited-receptor system to amplify a signaland generate a cellular response by rapid synthesis of small amounts ofPA. Uptake of di-unsaturated PA, which is less than about 0.1% of totalmembrane lipid mass, is sufficient to activate PLD activity. Thisquantity of PA is similar to that endogenously synthesized by LPAAT. ThePA-stimulated PLD acts upon PE, which should be localized to the innerleaflet of the cell membrane, enriched in PE relative to the outerleaflet. Therefore, the cellular inflammatory response to IL-1 ismediated by the pathway: IL-1R→PA→(PLD)→PE. Whereas a localized tissueresponse is: lysoPA→PI→PKC→(PLD)→PC. The PLD species are likely to bedifferent isozymes. The second messenger pathway whose activation isinhibited by the inventive compounds is not a PI-derived pathway anddoes not involve PKC in the time courses of inhibition. PKC is acutelyactivated by PI-derived DAG, but chronic activation (ie.,>30 minutes) ismaintained by PC-derived PA generated by PC-directed PLD. Therefore, thepathway inhibited by the inventive compounds is PE-directed and notPC-directed. Moreover, the PE-directed PLD favors substrates with sn-2long-chain unsaturation.

DAG and PA are upregulated in oncogenically transformed cells. Forexample, activating ras mutations result in increased generation of DAGupon stimulation with mitogens, although the sources of DAG differbetween experimental systems. In nontransformed renal mesangial cells,IL-1β stimulation increased PLA2 and LPAAT activation, resulting ingeneration of sn-2 unsaturated PA and subsequent hydrolysis to DAG byphosphatidate phosphohydrolase. The ras transformation in NIH/3T3 cellsupregulates serum-stimulated generation of DAG and PA. Particularspecies of DAG that is stimulated by serum is dioleoyl and of PA aredilinoleoyl and dioleoyl. This upregulation occurs over 4-12 hours andpretreatment of cells with an inventive compound, or PTX, blocksgeneration of these phospholipid second messengers. The inhibitionoccurs either through suppressing the generation of PA de novo fromlysoPA, or through inhibition of one or both arms of the Lands cycle.The coordinate increase of lysoPA in the setting of diminished PA/DAGproduction suggests inhibition of transacylation of a precursor lipid.Therefore, the ras transformation mediates an upregulation of PA throughindirect stimulation of PLA2 and/or LPAAT activity. The inventivecompounds inhibit the conversion of the upregulated lysoPA to PA andsubsequently block the phenotypic changes induced by PA/DAG in themembrane.

The ability of the inventive compounds to inhibit generation ofunsaturated phospholipids is mirrored by the ability of inventivecompounds to inhibit proliferation and tumorigenicity of ras-transformedcells in vitro and in vivo. PTX inhibits ras-transformed NIH3T3 cellsmore than parental cells. This inhibition is reversible and is notassociated with significant cytotoxicity.

Excessive or unregulated TNF (tumor necrosis factor) production isimplicated in mediating or exacerbating a number of diseases includingrheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, goutyarthritis and other arthritic conditions, sepsis, septic shock,endotoxic shock, gram negative sepsis, toxic shock syndrome, adultrespiratory distress syndrome, cerebral malaria, chronic pulmonaryinflammatory disease, silicosis, pulmonary sarcoidosis, bone resorptiondiseases, reperfusion injury, graft versus host reaction, allograftrejections, fever, myalgias due to infection such as influenza, cachexiasecondary to infection, AIDS or malignancy, AIDS, other viral infections(e.g., CMV, influenza, adenovirus, herpes family), keloid formation,scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.The inventive compounds or pharmaceutically acceptable salts thereof canbe used in the manufacture of a medicament for the prophylactic ortherapeutic treatment of any disease state in a human or other mammal,which is exacerbated or signaled through the present second messengercellular phospholipid-based signaling pathway and by excessive orunregulated production of "first messenger" inflammatory cytokines suchas TNF or IL-1. With regard to TNF first messenger signaling, there areseveral disease states in which excessive or unregulated TNF productionby monocytes/macrophages is implicated in exacerbating or causing thedisease. These include, but are not limited to, for example,neurodegenerative diseases such as Alzheimers disease, endotoxemia ortoxic shock syndrome (Tracey et al., Nature 330:662, 1987 and Hinshaw etal., Circ. Shock 30:279, 1990); cachexia (Dezube et al., Lancet 355:662,1990), and adult respiratory distress syndrome (Miller et al., Lancet2(8665):712, 1989). The inventive compounds may be used topically in thetreatment of prophylaxis of topical disease states mediated orexacerbated by excessive TNF or IL-1, such as viral infections (herpesor viral conjunctivitis), psoriasis, fungal or yeast infections(ringworm, athletes foot, vaginitis, dandruff, etc.) or otherdermatologic hyperproliferative disorders. High TNF levels have beenimplicated in acute malaria attacks (Grau et al., N. Engl. J Med.320:1585, 1989), chronic pulmonary inflammatory diseases such assilicosis and asbestosis (Piguet et al., Nature 344:245, 1990, andBissonnette et al., Inflammation 13:329, 1989), and reperfusion injury(Vedder et al., Proc. Natl. Acad. Sci. USA 87:2643, 1990).

The compounds of the invention can inhibit certain VEGF (vascularendothelial growth factor), FGF (fibroblast growth factor) and PDGF(platelet derived growth factor) effects in vivo, such as inhibition ofangiogenesis or restenosis. For example, Ferns et al., Science 253:1129,1991, have shown that neointimal smooth muscle chemotaxis andangioplasty are inhibited in rats using a neutralizing antibody to PDGF.Also, Jawien et al., J. Clin Invest. 89:507, 1992, have shown that PDGFpromotes smooth muscle migration and intimal thickening in a rat modelof balloon angioplasty. Inhibition of the PDGF-mediated effectsfollowing balloon angioplasty by the inventive compounds is thepharmacological rationale for using the inventive compounds astherapeutic agents to prevent restenosis. The inventive compounds alsoinhibit atherogenesis because increased levels of PDGF expressed bymacrophages are associated with all phases of atherogenesis (Ross etal., Science 248:1009, 1990). Further, many human tumors expresselevated levels of either PDGF, FGF, receptors for FGF or PDGF, ormutated cellular oncogenes highly homologous to these growth factors ortheir receptors. For example, such tumor cell lines include sarcoma celllines (Leveen et al., Int. J. Cancer 46:1066, 1990), metastatic melanomacells (Yamanishi et al., Cancer Res. 52:5024, 1992), and glial tumors(Fleming et al., Cancer Res. 52:4550, 1992).

The inventive compounds are also useful to raise the seizure threshold,to stabilize synapses against neurotoxins such as strychnine, topotentiate the effect of anti-Parkinson drugs such as L-dopa, topotentiate the effects of soporific compounds, to relieve motiondisorders resulting from administration of tranquilizers, and todiminish or prevent neuron overfiring associated with progressive neuraldeath following cerebral vascular events such as stroke. In addition,the compounds of the invention are useful in the treatment ofnorepinephrine-deficient depression and depressions associated with therelease of endogenous glucocorticoids, to prevent toxicity to thecentral nervous system of dexamethasone or methylprednisolone, and totreat chronic pain without addiction to the drug. Further, the compoundsof the invention are useful in the treatment of children with learningand attention deficits and generally improve memory in subjects withorganic deficits, including Alzheimer's patients.

Compounds of the Invention

The invention provides compounds that are useful therapeutic agents,inhibiting proinflammatory and neoplastic cellular signallingmechanisms. The inventive compounds and inventive pharmaceuticalcompositions thereof have the formula:

    CORE MOIETY --(R).sub.j

including resolved enantiomers and/or diastereomers, hydrates, salts,solvates and mixtures thereof, wherein j is an integer from one tothree, the core moiety is non-cyclic or cyclic (e.g. carbocyclic orheterocyclic) and R may be selected from among: hydrogen, halogen(preferably bromine, chlorine, fluorine and iodine), hydroxyl, amino,substituted or unsubstituted C.sub.(1-10) alkyl, C.sub.(2-10) alkenyl,carbocyclic or heterocyclic groups and formula I.

Preferred R substituents having a structure other than formula Iinclude, but are not limited to, 2-bromopropyl, 4-chloropentyl,cyclohexyl, cyclopentyl, 3-dimethylaminobutyl, ethyl, hexyl,2-hydroxyethyl, 5-hydroxyhexyl, 3-hydroxy-n-butyl, 3-hydroxypropyl,isobutyl, isopropyl, 2-methoxyethyl, 4-methoxy-n-butyl, methyl, n-butyl,n-propyl, phenyl, t-butyl and the like. Particularly preferred R havinga structure other than formula I are ethyl, methyl, or hydrogen.

The inventive compounds have at least one R of the following formula I:##STR4## wherein: one or two p are the integer one, otherwise p is two;

n is an integer from three to twenty.

R₁ is selected from among substituted and unsubstituted CH₂ ; NR₃ (R₃being hydrogen, substituted or unsubstituted C.sub.(1-20) alkyl,C.sub.(1-20) alkoxyl, C.sub.(2-20) alkenyl or C.sub.(1-20) hydroxyalkyl,or carbocyclic or heterocyclic group); O; --CHR₄ O-- (R₄ beingsubstituted or unsubstituted C.sub.(1-20) alkyl, C.sub.(1-20) alkoxyl,C.sub.(2-20) alkenyl, C.sub.(1-20) hydroxyalkyl or R₂ and R₄ join toform a substituted or unsubstituted heterocycle having four to sevenring atoms, the ether group --O-- of --CHR₄ O-- being a member of theheterocycle); R₂ is hydrogen, halogen, substituted or unsubstitutedC.sub.(1-10) alkyl, C.sub.(1-10) alkoxyl, C.sub.(2-10) alkenyl,C.sub.(1-10) hydroxyalkyl, --A(R₅)_(m) (A being N or O, m being one ortwo and R₅ being hydrogen, a substituted or unsubstituted carbocyclic orheterocyclic group having at least one four- to seven-membered ring;substituted or unsubstituted C.sub.(1-10) alkyl, C.sub.(1-10) alkoxyl,C.sub.(2-10) alkenyl or C.sub.(1-10) hydroxyalkyl).

In the inventive compounds, at least one of R₁ is NR₃, O or --CHR₄ O--,or R₂ is --A(R₅)_(m). In addition when the core moiety is xanthine, R₂is --A(R₅)_(m), A is --O-- and R₅ is hydrogen or C.sub.(1-10) alkyl, nis not less than four. Optionally, (CH₂)_(n) may 1) be substituted by ahalogen, hydroxide, substituted or unsubstituted C.sub.(1-10) alkyl,C.sub.(2-10) alkenyl, C.sub.(1-10) alkoxyl, C.sub.(1-10) acyloxy,C.sub.(1-10) oxyalkyl, carbocyclic or heterocyclic group; 2) have one ortwo unsaturated bonds (preferably in a cis configuration); or 3) beinterrupted by at least one oxygen atom.

Preferably, n is an integer from about three to about eighteen, morepreferably, an integer from about four to about ten. In especiallypreferred compounds, R₁ is NR₃, R₃ is C.sub.(1-20) alkyl, and R₂ isC.sub.(1-10) alkyl or hydroxyalkyl. Even more preferably, (CH₂)_(n) issubstituted by an hydroxide, a C.sub.(1-10) alkyl or C.sub.(1-10)acyloxy. Other preferred embodiments may include, but are not limitedto, compounds in which R₁ is O, R₂ is C.sub.(1-10) alkyl, C.sub.(2-10)alkenyl or C.sub.(1-10) alkoxyl and (CH₂)_(n) is substituted by ahalo-substituted C.sub.(1-10) alkyl, or unsubstituted C.sub.(2-10)alkenyl or C.sub.(1-10) alkoxyl.

Although other possible substituents are within the scope of theinventive compounds, representative substituents, when R, R₂ or R₅ is asubstituted C.sub.(1-10) alkyl, C.sub.(2-10) alkoxyl, C.sub.(2-10)alkenyl or C.sub.(1-10) hydroxyalkyl, may be: amide, primary, secondaryand tertiary amine, C.sub.(2-8) alkenyl, C.sub.(1-8) alkyl (including,e.g., branched and unbranched alkyl or alkenyl groups), C.sub.(1-8)alkoxyl, C.sub.(1-8) hydroxyalkyl, azide, carbonate, carbonyl,carboxylic acid, cyanide, C.sub.(1-8) haloalkyl (including, e.g., mono-,di- and tri-haloalkyl substituents, such as trihalomethyl), isocyanate,isothiocyanate, phosphate, phosphonate, primary, secondary or tertiaryalcohol (including, e.g., any one of various diols, methanol, butanol,1-cyclopentanol, ethanol, 2-ethyl-3-methyl-1-propanol, pentanol,propanol, and methylcyclohexanol), sulfonate, sulfone, sulfoxide,thioamide, thiocarbonate, thioester, thiolester, thiol, thiourea andurea.

The above-listed, substituents are also representative of substituentswhen R₃ or R₄ is a substituted C.sub.(1-20) alkyl, C.sub.(1-20) alkoxyl,C.sub.(2-20) alkenyl or C.sub.(1-20) hydroxyalkyl; R, R₃ or R₅ is asubstituted carbocyclic or heterocyclic group; or R₁ is a substitutedCH₂.

Representative R, R₃ or R₅ carbocyclic or heterocyclic groups may be,but are not limited to: anthracene, bicyclo[4.4.0]decane,bicyclo[2.2.1]heptane, bicyclo[3.2.0]heptane, bicyclo[4.1.0]heptane,bicyclo[2.2.1]hexane, bicyclo[4.3.0]nonane, bicyclo[2.2.2]octane,biphenyl, cyclopentadiene, cyclopentane, cyclobutane, cyclobutene,cycloheptane, cyclohexane, cyclooctane and cyclopropane,1,2-diphenylethane, fluorene, indene, phenyl, quinone, terphenyl,napthalene, phenanthrene, terphenyl, toluene, xylene, azetidine,benzofuran, benzothiophene, carbazole, furan, glutarimide, indole,isoquinoline, lactam, lactone, oxazole, oxetane, oxirane, phthalimide,piperidine, pyrrolidine, pyran, pyridine, pyrrole, quinoline,tetrahydrofuran, tetrahydropyran, tetrahydrothiophene, thiophene,thymine, derivatives thereof and the like. Due primarily to availabilityand ease of synthesis, more preferred cyclic (carbocyclic orheterocyclic) groups include, but are not limited to, less complex ringsystems, such as, for example, cyclopentane and cyclohexane,cyclopentadiene, phenyl, indene, toluene, xylene, furan, indole, thymineand xanthine.

A non-cyclic core moiety may include, but is not limited to, forexample, acetamide, amide, amine, amino acid (one or two), carboxide,ester, terminal halogen or hydrogen atom, hydroxide, glutaric acid,glycine derivative, ketone, phosphate, phosphonate, sulfate, sulfonate,sulfone, sulfoxide, simple ionic functional group, thiol, thiolester orthe like. Exemplary core moiety amino acids may include, but is limitedto, one or more of the following: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine. The non-cyclic core moietymay preferably be an amide, carboxyl ester, carboxide, hydrogen,hydroxide or a dipeptide comprising two amino acids selected from theforegoing exemplary list. A non-cyclic, halogen-core moiety may be, forexample, bromine, chlorine, fluorine or iodine.

A cyclic core may be at least one five- to seven-member,non-heterocyclic (i.e. carbocyclic) ring or a heterocycle. The at leastone five- to seven-membered cyclic core may preferably have from one tothree, five- to six-membered ring structures in a predominantly planarconfiguration. An exemplary, non-heterocyclic ring core moiety may beselected from the group consisting of substituted or unsubstitutedbenzene; biphenyl; cyclohexane; cyclohexanedione; cyclopentanedione;napthlalene; phenol; quinone; salicylic acid; stilbene andtricyclododecane.

Although other heterocyclic cores are within the scope of the invention,the following representatives are preferred: substituted orunsubstituted barbituric acid; benzamide; lactam; glutarimide;homophthalimide; hydrophthalimide; imidazole; imidazole amide;indomethacin; isocarbostyril; lumazine; N-alkylheterocyclic;N-heterocyclic; pteridine; pthalimide; piperidine; pyridine; pyrimidine;pyrrole amide; quaternized N-heterocyclic; quinolizinedione;quinazolinone; quinoline; recorsinol; succinimide; theobromine; thymine;triazine; uric acid; uracil; vitamins A, E or K; or xanthine.

Representative substituents for the non-heterocyclic (i.e., carbocyclic)or heterocyclic core moieties include, for example, amide, primary,secondary and tertiary amine, C.sub.(2-8) alkenyl, C.sub.(1-8) alkyl(including, e.g., branched and unbranched alkyl or alkenyl groups),C.sub.(1-8) alkoxyalkyl, azide, carbonate, carbonyl, carboxylic acid,cyanide, C.sub.(1-8) haloalkyl (including, e.g., mono-, di- andtri-haloalkyl substituents, such as trihalomethyl), isocyanate,isothiocyanate, phosphate, phosphonate, primary, secondary or tertiaryalcohol (including, e.g., any one of various diols, methanol, butanol,1-cyclopentanol, ethanol, 2-ethyl-3-methyl-1-propanol, pentanol,propanol, and methylcyclohexanol), sulfonate, sulfone, sulfoxide,thioamide, thiocarbonate, thioester, thiolester, thiol, thiourea andurea.

Preferred non-heterocyclic ring cores include, but are not limited to,substituted or unsubstituted 1,3-cyclohexanedione,1,3-cyclopentanedione; 1,3-dihydroxynaphthalene; or orthophenol.

Preferred heterocyclic cores include, but are not limited to,substituted or unsubstituted 3,7-dimethylxanthine, glutarimide,3-methyl-7-pivoloylxanthine, methylthymine, methyluracil,3-methylxanthine, tetrahydrophthalimide, thymine, uracil and xanthine,most preferably methyl-substituted xanthine. Exemplary preferred coresinclude, but are not limited to: C.sub.(1-6) alkyl-substituted thymine;C.sub.(1-6) alkyl-substituted uracil; 1,3-dihydroxynapthalene;3,3-dimethylglutarimide; dihydrothymine;2,4-dioxohexahydro-1,3,5-tetrazine; hexahydrophthalimide;homophthalimide; 2-hydroxypyridine; β-ionone as vitamin Amethylbarbituric acid; 2,6,6-methyl-1-cyclohexene-1-acetaldehyde asvitamin A; methyldihydroxypyrazolopyrimidine, specifically,1,3-dimethyldihydroxypyrazolo[4,3-d]pyrimidine;1-methyl-5,6-dihydrouracil;1,7-dimethylxanthine, 3,7-dimethylxanthine; 7-methylhypoxanthine;1-methyllumazine; 3-methyl-7-methylpivaloylxanthine;methylpyrrolopyrimidine; 1-methylpyrrolo [2,3-d] pyrimidine;1-methyl-2,4(1H,3H)-quinolizinedione (1-methylbenzoyleneurea);methylthymine; 1-methyluracil; 3-methylxanthine; orotic acid;prostacyclin; 1-pyrrole amides; 2-pyrrole amides; 3-pyrrole amides;quinazolin-4(3H)-one; 1,2,3,4-tetrahydroisoquinoline;tetrahydrophthalimide; sulindac; uracil fused to naphthalene; 5- and/or6-position substituted uracils (such as, for example, 5-bromouracil);tetralone to vitamin K; and 8-substituted xanthines (having substituentssuch as N or S).

Preferably, R is bonded to a nitrogen of the core moiety, if present,most preferably to the nitrogen of a glutarimide, methylthymine,thymine, uracil or xanthine core. In representative, preferredcompounds, R having formula I may be bonded to an N₁ nitrogen ofglutarimide; N₁ nitrogen of xanthine (and N₃ and N₇ xanthine nitrogensmay be independently substituted by a member selected from the groupconsisting of hydrogen, C.sub.(1-6) alkyl, fluoro, chloro and amino); N₃nitrogen of methylthymine; or N₁ nitrogen of uracil. Alternatively, Rhaving formula I may be bonded to N₁ and N₃ xanthine nitrogens and N₇xanthine nitrogen is substituted by a member selected from the groupconsisting of hydrogen, methyl, fluoro, chloro and amino.Representative, preferred inventive compounds are compounds of formulasII, III and IV: ##STR5## wherein R is defined above.

The invention also provides a pharmaceutical composition. Pharmaceuticalcompositions of the inventive compounds comprise a pharmaceuticalcarrier or diluent and some amount of an inventive compound. Thecompound may be present in an amount to effect a physiological response,or it may be present in a lesser amount such that the user will need totake two or more units of the composition to effect the treatmentintended. These compositions may be made up as a solid, liquid or in agaseous form. Or one of these three forms may be transformed to anotherat the time of being administered such as when a solid is delivered byaerosol means, or when a liquid is delivered as a spray or aerosol.

The nature of the composition and the pharmaceutical carrier or diluentwill, of course, depend upon the intended route of administration, forexample, parenterally, topically, orally or by inhalation for treatmentof a patient with disease symptoms. For topical administration, thepharmaceutical composition will be in the form of a cream, ointment,liniment, lotion, paste, aerosol or drop suitable for administration tothe skin, eye, ear, lung or nose. For parenteral administration, thepharmaceutical composition will be in the form of a sterile injectableliquid. For oral administration, the pharmaceutical composition will bein the form of a tablet, capsule, powder, pellet, atroche, lozenge,syrup, liquid, emulsion or aqueous or non-aqueous liquid suspension.

The invention includes a method for treating an individual having avariety of diseases. The disease is characterized by or can be treatedby inhibiting an immune response or a cellular response to external orin situ primary stimuli. Treatment of the disease states involvesmediating the cellular response through a specific phospholipid-basedsecond messenger pathway acting adjacent to a cell membrane innerleaflet. The second messenger pathway is activated in response tovarious noxious or proliferative stimuli, characteristic of diseasestates treatable using the inventive compounds or pharmaceuticalcompositions thereof. The inventive compounds are active by inhibitingvarious enzymes of this phospholipid second messenger pathway. The coremoiety component of the inventive composition might serve to anchor thecompound to an inner leaflet of a cell's plasma membrane allowing an "R"moiety of the inventive compound to interact with or inhibit an enzymeinvolved in phospholipid metabolism, usually leading to cellularaccumulation of specific PA (phosphatidic acid) species.

More specifically, the invention includes methods for treating orpreventing clinical symptoms of various disease states or reducingtoxicity of other treatments by inhibiting cellular signaling through asecond messenger pathway involving signaling through phosphatidic acidand through glycan phosphatidylinostinol (Gly PI).

Illustrative examples of compounds of the present invention include, butare not limited to, the following: ##STR6## Method of Making theInventive Compounds

The invention also provides a process for preparing the inventivecompounds. The inventive process utilizes starting materials availableto skilled artisans, whether commercially supplied or prepared fromother materials commercially available. In addition, some, selectedstarting materials and intermediates available for use in the inventiveprocess and a corresponding method of synthesis for these selectedstarting materials are disclosed in U.S. patent applications, Ser. Nos.08/152,650 now U.S. Pat. No. 5,837,703 issued Nov. 17, 1998, and08/164,081 now U.S. Pat. No. 5,470,878 issued Nov. 28, 1995, filed Nov.12, 1993 and Dec. 8, 1993, respectively, the disclosures of which areincorporated in their entirety herein by reference.

The inventive carboxylic acid-, ester- and amide-substituted compoundsof the invention may be prepared by the following general process.Specific, non-limiting examples of synthetic protocols for preparingexemplary compounds of the invention are set forth in the examples whichfollow.

In a method according to the invention, a compound containing a desiredcore (intended as a "core moiety" in the inventive compound) undergoes areaction to produce an anion. Then, the resulting anion may besubsequently reacted with a suitable, substituted ester having at leastone other functional group to displace a targeted functional group onthe ester, thereby obtaining a compound according to the invention.

In a preliminary reaction, a predetermined amount of a core-containingcompound is reacted with a base, a solvent and the suitable substitutedester to obtain an ester product. Again, the substituted ester has atleast one functional group which may be substituted in a displacementreaction by the desired core-containing compound.

Preferred bases include, but are not limited to, sodium hydride, sodiumamide, sodium alkoxide, lithium hydride, potassium hydride, lithiumamide, sodium amide and potassium amide. An especially preferred base issodium hydride. Preferred solvents may be dimethylsulfoxide,dimethylformamide, or an alcohol. An alcohol may be chosen from amongmethanol, ethanol or isopropanol. Any substituted ester comprising achain structure of the inventive compounds may be used in thispreliminary reaction, as long as a functional group is present fordisplacement. Preferred esters may be substituted esters and may be, butare not limited to, halo-substituted esters.

These ester products, which have a composite structure of a core-moietyand ester-containing side chain may then subsequently be converted to aninventive compound having a carboxylic acid-substituted side chain.

In this process, the ester product is reacted with an ester-hydrolyzingagent to obtain an inventive compound having a carboxylicacid-substituted side chain. Representative ester-hydrolyzing agentsuseful in preparing inventive carboxylic acid-containing inventivecompounds may be potassium hydroxide or sodium hydroxide in water,although other ester-hydrolyzing agents are within the scope of theinventive process.

In a halogenation reaction, the carboxylic acid-containing compoundabove may be reacted with a halogenating agent to obtain an intermediatehaving a carboxylic acid halide functional group. Although other agentsare within the scope of the inventive method, halogenating agents may bechosen from among thionyl chloride, phosphorus trichloride, phosphoruspentachloride, phosphorus oxychloride, thionyl bromide and the like.

Once the intermediate prepared in the step above, containing acarboxylic acid halide functional group is isolated, it is then bereacted with an amine to obtain a corresponding amide-containinginventive compound. In this reaction, the amine compound will contributeto a portion of the final structural configuration of the inventiveamide-containing compounds.

Alternatively, a compound containing a desired core may be reacted witha base and substituted-olefin, producing an intermediate olefinicproduct. The substituted olefin starting material will have a targetfunctional group which will be displaced by an anion of thecore-containing compound. In this reaction, a predetermined amount of acore-containing compound is reacted with a suitable base, a solvent anda substituted olefin. Again, the substituted olefin has at least onefunctional group for displacement.

Preferred bases include, but are not limited to, sodium hydride, sodiumamide, sodium alkoxide, lithium hydride, potassium hydride, lithiumamide, sodium amide, potassium amide and sodium hydride. Preferredsolvents may be dimethylsulfoxide, dimethylformamide, or an alcohol suchas, for example, methanol, ethanol or isopropanol. Any substitutedolefin comprising a chain structure of the inventive compounds may beused in the preliminary reaction according to the invention. Preferredolefins may be substituted olefins. Preferred substituted olefinsinclude, but are not limited to halo-substituted olefins.

By reacting the intermediate olefinic product previously obtained withan oxidizing agent, a diol is prepared from the olefinic product.Preferred oxidizing agents include, but are not limited to, osmiumtetroxide. Preferred oxidizing agents, such as osmium tetroxide mayrequire a catalytic amount of the oxidizing agent in the presence of aregenerating agent. Representative regenerating agents may be4-methylmorpholine-N-oxide and trimethylamine-N-oxide. An especiallypreferred regenerating agent is 4-methylmorpholine-N-oxide. In asubsequent halogenation reaction, the resulting diol is converted to aninventive compound using a halogenating agent in the presence of anorganic acid. Exemplary halogenating agents include, but are not limitedto, hydrogen bromide and hydrogen chloride. Preferred organic acids maybe acetic acid and propionic acid.

Also, inventive amide- and ester-substituted compounds according to theinvention may also be prepared by reacting a compound containing atleast one of an alcohol or amine functional group with a substitutedacyl halide or carboxylic acid anhydride. The compound containing atleast one alcohol or amine also has as a structural component a coremoiety corresponding to a core moiety of the inventive compounds.Starting materials may be obtained commercially or by synthesis fromother materials which are commercially available. Some amino alcoholcompounds may also be prepared as disclosed in the above-identifiedcopending U.S. patent applications.

A schematic representation of an inventive process for preparing anamide-substituted inventive compound is illustrated as follows: ##STR7##

inventive amide-containing compound

Uses of the Invention Compounds and Pharmaceutical Formulations

The inventive compounds provide a method for maintaining homeostasis incells contacted by primary stimuli by mitigating the effects of theseprimary stimuli on the secondary signaling pathways invoked withinseconds of a primary stimulus. For example, administration of aninventive compound in vivo or ex vivo provides a method to modifycellular behavior, the method comprising contacting cells (in vivo or exvivo), whose behavior is to be modified, with an effective amount of aninventive compound or a pharmaceutical composition thereof. The methodis a method to: (1) inhibit proliferation of tumor cells, being; (2)suppress activation of T-cells by antigen or IL-2 stimulation being; (3)suppress activation of monocyte/macrophage cells by endotoxin, TNF, IL-1or GM-CSF stimulation, being; (4) suppress antibody production ofB-cells in response to an antigen, IL-4 or CD40 ligand, being; (5)inhibit the proliferation of smooth muscle cells in response to growthfactors capable of stimulating said proliferation, being; (6) lowersystemic vascular resistance conferred by endothelial cells, being; (7)lower systemic vascular resistance induced by endothelial cells, being;(8) lower expression of adhesion molecules induced by enhancers thereof,being; (9) suppress the activation of T-cells and macrophages by HIV,being; (10) inhibit the proliferation of kidney mesangial cells inresponse to stimulation by IL-1 and/or MIP-1α and/or PDGF and/or FGF,being; (11) enhance the resistance of kidney glomerular or tubular cellsto cyclosporin A or amphotericin B, being; (12) prevent the release ofMIP-1 by IL-1, TNF, or endotoxin stimulated monocytes and macrophages;(13) prevent the release of platelet activating factor by IL-1, TNF, orendotoxin treated megakaryocytes, fibroblastic cells, and macrophages;(14) prevent the down-regulation of receptors for cytokines inTNF-treated hematopoietic progenitor cells, being; (15) suppress theproduction of metalloproteases in IL-1-stimulated or TNF-stimulatedglomerular epithelial cells or synovial cells, being; (16) enhance theresistance of gastrointestinal or pulmonary epithelial cells tocytotoxic drugs or radiation, being; (17) enhance the antitumor effectof a non-alkylating antitumor agent, being; (18) to inhibit theproduction of osteoclast activating factor in response to IL-1, being;(19) inhibit degranulation in response to IgE, being; (20) enhance therelease of adrenergic neural transmitters, dopamine, norepinephrine, orepinephrine, or the neurotransmitter, acetylcholine, being; (21)modulate the post-synaptic "slow current" effects of the adrenergicneurotransmitters dopamine, epinephrine, or norepinephrine, or theneurotransmitter acetylcholine, being; (22) suppress signaling byneurotransmitters including acetyl choline, leuenkephalin and serotonin;or (23) increase seizure threshold.

Indications useful for administering compounds of the invention include,but are not limited to: the presence of a tumor burden, ahormone-related disorder, a neurological disorder, an autoimmunedisease, inflammation, restenosis, coronary artery disease,atherosclerosis, hypertension, unwanted immune response (such asallograft reactions), viral infection, nephritis, mucositis, and variousallergic responses. Allergic responses include, but are not limited to,acute allergic response and thus rhinorrhea, sinus drainage, diffusetissue edema, and generalized pruritus. As well as the following, otherchronic allergic responses include, but are not limited to, dizziness,diarrhea, tissue hyperemia, and lacrimal swelling with localizedlymphocyte infiltration. Allergic reactions are also associated withleukotriene release and the distal effects thereof, including asthmaticsymptoms (e.g., development of airway obstruction, a decrease in FEV1,changes in vital capacity, and extensive mucus production).

Other suitable subjects for the administration of compounds of theinvention, include patients: being administered other cytotoxic agentsfor the treatment of tumors, such as chemotherapeutic agents orirradiation therapy; suffering from neoplasias generally, whether or nototherwise treated including acute and chronic myelogenous leukemia,hairy cell leukemia, lymphomas, megakaryocytic leukemia, and the like;disease states caused by bacterial, fungal, protozoal, or viralinfection; exhibiting unwanted smooth muscle cell proliferation in theform of, for example, restenosis, such as patients undergoing cardiacsurgery; afflicted with autoimmune diseases, thus requiring deactivationof T and B cells, and having neurological disorders.

The compounds of the invention further are able to decrease enhancedlevels of a relevant PA and DAG resulting from stimulation ofsynaptosomes with acetylcholine and/or epinephrine. This suggests thatthe effects of the compounds of the invention are to both enhance therelease of inhibitory neural transmitters such as dopamine, and tomodulate the distal "slow current" effects of such neurotransmitters.

Thus, the drugs of the invention are also useful to raise the seizurethreshold, to stabilize synapses against neurotoxins such as strychnine,to potentiate the effect of anti-Parkinson drugs such as L-dopa, topotentiate the effects of soporific compounds, to relieve motiondisorders resulting from administration of tranquilizers, and todiminish or prevent neuron overfiring associated with progressive neuraldeath following cerebral vascular events such as stroke. In addition,the compounds of the invention are useful in the treatment ofnorepinephrine-deficient depression and depressions associated with therelease of endogenous glucocorticoids, to prevent the toxicity to thecentral nervous system of dexamethasone or methylprednisolone, and totreat chronic pain without addiction to the drug. Further, the compoundsof the invention are useful in the treatment of children with learningand attention deficits and generally improve memory in subjects withorganic deficits, including Alzheimer's patients.

While dosage values will vary, therapeutic efficacy is achieved when thecompounds of the invention are administered to a human subject requiringsuch treatment as an effective oral, parenteral, or intravenoussublethal dose of about 50 mg to about 5000 mg per day, depending uponthe weight of the patient. A particularly preferred regimen for use intreating leukemia is 4-50 mg/kg body weight. It is to be understood,however, that for any particular subject, specific dosage regimensshould be adjusted to the individual's need and to the professionaljudgment of the person administering or supervising the administrationof the inventive compounds.

Pharmaceutical Formulations

A suitable formulation will depend on the nature of the disorder to betreated, the nature of the medicament chosen, and the judgment of theattending physician. In general, the inventive compounds are formulatedeither for injection or oral administration, although other modes ofadministration such as transmucosal or transdermal routes may beemployed. Suitable formulations for these compounds can be found, forexample, in Remington's Pharmaceutical Sciences (latest edition), MackPublishing Company, Easton, Pa.

The inventive compounds and their pharmaceutically acceptable salts canbe employed in a wide variety of pharmaceutical forms. The preparationof a pharmaceutically acceptable salt will be determined by the chemicalnature of the compound itself, and can be prepared by conventionaltechniques readily available. Thus, if a solid carrier is used, thepreparation can be tableted, placed in a hard gelatin capsule in powderor pellet form or in the form of a troche or lozenge. The amount ofsolid carrier will vary widely but preferably will be from about 25 mgto about 1 gram, wherein the amount of inventive compound per dose willvary from about 25 mg to about 1 gram for an adult. When a liquidcarrier is used, the preparation will be in the form of a syrup,emulsion, soft gelatin capsule, sterile injectable liquid such as anampule or nonaqueous liquid suspension. Where the inventive compositionis in the form of a capsule, any routine encapsulation is suitable, forexample, using the aforementioned carriers in a hard gelatin capsuleshell. Where the composition is in the form of a soft gelatin shellcapsule, any pharmaceutical carrier routinely used for preparingdispersions of suspensions may be considered, for example, aqueous gums,celluloses, silicates or oils and are incorporated in a soft gelatincapsule shell. A syrup formulation will generally consist of asuspension or solution of the compound or salt thereof in a liquidcarrier (e.g., ethanol, polyethylene glycol, coconut oil, glycerine orwater) with a flavor or coloring agent.

The amount of inventive compound required for therapeutic effect ontopical administration will, of course, vary with the compound chosen,the nature and severity of the disease and the discretion of thetreatment provider. Parenteral includes, but is not limited to,intravenous, intramuscular, subcutaneous, intranasal, intrarectal,intravaginal or intraperitoneal administration. Appropriate dosage formsfor such administration may be prepared by conventional techniques. Atypical parenteral composition consists of a solution or suspension ofthe inventive compound or a salt thereof in a sterile or non-aqueouscarrier, optionally containing a parenterally acceptable oil, forexample polyethylene glycol, polyvinylpyrrolidone, lecithin, arachisoil, or sesame oil. The daily dosage for treatment of sepsis or anothersevere inflammatory condition via parenteral administration is suitablefrom about 0.001 mg/kg to about 40 mg/kg, preferably from about 0.01mg/kg to about 20 mg/kg of an inventive compound or a pharmaceuticallyacceptable salt thereof calculated as the free base.

The inventive compounds may be administered orally. The daily dosageregimen for oral administration is suitably from about 0.1 mg/kg toabout 1000 mg/kg per day. For administration the dosage is suitably fromabout 0.001 mg/kg to about 40 mg/kg of the inventive compound or apharmaceutically acceptable salt thereof, calculated as the free base.The active ingredient may be administered from 1 to 6 times a day,sufficient to exhibit activity.

The inventive compounds may be administered by inhalation (e.g.,intranasal or oral). Appropriate dosage forms include, but are notlimited to, an aerosol or a metered dose inhaler, as prepared byconventional techniques. The daily dosage is suitably from about 0.001mg/kg to about 40 mg/kg of the inventive compound or a pharmaceuticallyacceptable salt thereof, calculated as the free base. Typical compoundsfor inhalation are in the form of a solution, suspension or emulsionthat may be administered as a dry powder or in the form of an aerosolusing a conventional propellant.

The invention is illustrated by the following examples which should notbe regarded as limiting the invention in any way.

EXAMPLE 1

This example is a synthesis for inventive compound no. 1527 (see abovefor chemical name and structure). A mixture of theobromine (1.0 g, 5.5mmol, available from Sigma) and a solution (20 ml) of 50% sodium hydridein oil (264 mg, 5.5 mmol) in dimethylsulfoxide was stirred for 50minutes, followed by addition of 6-bromo-1-hexanol (1.0 g, 5.5 mmol,available from Aldrich). After stirring for 18 hours, the solution wastreated with 50 ml of water and then extracted with two 25 ml aliquotsof hexanes. The aqueous phase was extracted with three 35 ml aliquots of25% ethanol-dichloromethane. The combined ethanol-dichloromethaneextracts were dried over magnesium sulfate and then the solvents wereevaporated under vacuum. The remaining dimethylsulfoxide was removed bydistillation under full pump vacuum, producing 1.4 g of a white powder,1-(6-hydroxyhexyl)-3,7-dimethylxanthine (5.0 mmol, 91% yield).

A solution (5 ml) of chloroacetyl chloride (339 mg; 3 mmol) indichloromethane was added dropwise at 0° C. to a solution (5 ml) of1-(6-hydroxyhexyl)-3,7-dimethylxanthine (560 mg; 2 mmol) andtriethylamine (607.2 mg; 6 mmol) in dichloromethane. The reaction wasslowly warmed to room temperature and stirred overnight. The reactionwas quenched with saturated sodium bicarbonate solution (5 ml) andextracted with three 50 ml aliquots of dichloromethane. The combinedorganic extracts were washed with 1% dilute hydrogen chloride (15 ml),followed by water (15 ml) and finally with brine solution (15 ml), driedover anhydrous magnesium sulfate and concentrated under reducedpressure. A crude product obtained was further purified by flashchromatography over silica gel using a 20% hexane/ethyl acetate eluant,resulting in 296 mg of compound no. 1527 (50.1% yield).

EXAMPLE 2

Theobromine (11.9 g, 66 mmol, available from Sigma) was added to amixture of bromohexene (10.7 g, 66 mmol, available from Aldrich) andsodium hydride (1.58 g, 66 mmol) in dimethylsulfoxide (100 ml) and theresulting mixture stirred for 43 hours. The solution was treated withwater (200 ml) and then extracted with three 80 ml aliquots ofdichloromethane. The combined extracts were washed with three 100 mlaliquots of water and dried over magnesium sulfate. The solvent wasevaporated under vacuum, leaving 17 g of a white powder,1-(5-hexenyl)-3,7-dimethylxanthine (65 mmol, 98% yield).

Six drops of 2.5% osmium tetraoxide in t-butanol were added to a mixtureof 1-(5-hexenyl)-3,7-dimethylxanthine (1.07 g, 4.1 mmol), as preparedabove and N-methylmorpholine-N-oxide (1.44 g, 12.3 mmol) in water (20ml) and acetone (10 ml). After stirring the resulting mixture for 48hours, the mixture was treated with 20% aqueous sodium dithionitesolution (20 ml). After 2 minutes, the mixture was extracted with three30 ml aliquots of a 25% ethanol-dichloromethane solution. The combinedextracts were dried over magnesium sulfate and the solvent wasevaporated under vacuum, leaving 750 mg of a white powder,1-(5,6-dihydroxyhexyl)-3,7-dimethylxanthine (2.53 mmol, 62% yield).

A solution of 1-(5,6-dihydroxyhexyl)-3,7-dimethylxanthine (0.50 g, 1.7mmol) and 1,1'-carbonyldiimidazole (1.10 g, 6.8 mmol) was refluxed for20 hours. Water (30 ml) was added and the mixture was extracted withthree 50 ml aliquots of dichloromethane. The combined organic layerswere washed with two 30 ml aliquots of water and dried over sodiumsulfate. The solvent was removed under vacuum. A residue was furtherpurified by chromatography over silica using an ethyl acetate-10%ethanol eluant, yielding 180 mg of compound no. 1578 (33% yield).

EXAMPLE 3

Methanesulfonyl chloride (2.20 g, 1.5 ml, 19.2 mmol) was added to asolution (100 ml) of 9-decene-1-ol (3.00 g, 19.2 mmol, available fromAldrich) in dichloromethane at 0° C., followed by addition oftriethylamine (2.91 g, 28.8 mmol). After stirring was continued for 15minutes at 0° C., the reaction was allowed to warm to room temperature.After 2 hours, the reaction mixture was poured into water (100 ml) andextracted with three 60 ml aliquots of dichloromethane. The combinedorganic portions were dried over sodium sulfate and the solvent wasevaporated under vacuum, leaving a yellow oil mesylate (4.52 g, 100%),which was used without further purification.

Theobromine (3.45 g, 19.2 mmol) was added to a suspension (30 ml) ofsodium hydride (461 mg, 19.2 mmol) in dimethylsulfoxide. After 15minutes, the 9-decenylmesylate (2.25 g, 11 mmol) was added and thereaction stirred for 18 hours at 25° C., then for 40 minutes at 100° C.The mixture was then poured into water (100 ml) and extracted with three50 ml aliquots of dichloromethane. The combined organic portions werewashed with saturated salt solution (60 ml) and dried over magnesiumsulfate. Evaporating the solvent under vacuum left a white solidresidue. Recrystallization of the residue in ether produced 3.40 g of1-(9-decenyl)-3,7-dimethylxanthine (56% yield).1-(9-Decenyl)-3,7-dimethylxanthine (3.2 g, 10.1 mmol),4-methylmorpholine-N-oxide (1.41 g, 12 mmol), 3 drops of 2.5% osmiumtetroxide solution in t-butanol, acetone (40 ml) and water (10 ml) werestirred for 24 hours. Following addition of 5 ml of a saturated solutionof sodium dithionite and a further 15 minutes of stirring, the reactionmixture was extracted with four 50 ml aliquots of 25%ethanol/dichloromethane. The combined organic portions were dried oversodium sulfate. Evaporating the solvents left a white solid residue,which upon recrystallization in ethanol produced 3.3 g of1-(9,10-dihydroxydecyl)-3,7-dimethylxanthine (93% yield).1-(9,10-Dihydroxydecyl)-3,7-dimethylxanthine (2.11 g, 6 mmol), preparedabove, was stirred with hydrogen bromide (3.58 ml, 4.85 g of a 30%solution in acetic acid, 18 mmol) for 90 minutes. The mixture was thenadded to a flask containing 40 ml aqueous sodium bicarbonate solution (5g) and 50 ml dichloromethane. After 10 minutes of vigorous stirring thelayers were separated and the aqueous portion washed with two 50 mlaliquots of dichloromethane. The organic portions were combined, driedover sodium sulfate, and evaporating the solvent produced 2.72 g of ayellow oil, inventive compound no. 1583 (100% yield).

EXAMPLE 4

Sodium hydride (343 mg, 14 mmol) was added to a stirring solution of1-methylthymine (2.00 g, 14 mmol) in dimethylsulfoxide (40 ml). After 15minutes, 9-bromo-1-nonene (2.93 g, 14 mmol, available from Alfebro) wasadded and the resulting mixture stirred for 20 hours. The reaction waspoured into water (40 ml) and extracted with three 50 ml aliquots ofdichloromethane. The organic layers were combined, washed with water (40ml) and saturated aqueous salt solution (20 ml). After drying the washedorganic layers over sodium sulfate, the solvent was evaporated, leavinga colorless oil, 3-(8-nonenyl)-1-methylthymine, which solidified uponstanding (2.76 g, 73% yield).

A solution of 3-(8-nonenyl)-1-methylthymine (2.63 g, 9.9 mmol), preparedabove, 4-methylmorpholine-N oxide (1.39 g, 12 mmol), and potassiumosmate (IV) dihydrate (7 mg, 2×10⁻⁵ mol) in acetone (20 ml) and water(10 ml) was stirred for 18 hours. After addition of a saturated aqueoussolution of sodium hydrosulfite (10 ml) and 15 minutes of stirring, thereaction mixture was extracted with dichloromethane (50 ml) and with two50 ml aliquots of dichloromethane/20% methanol. The combined organiclayers were washed with water (15 ml) and saturated aqueous saltsolution (15 ml), and then dried over sodium sulfate. The solvent wasevaporated under vacuum, leaving a white solid residue.Recrystallization of the solid in ethanol yielded 2.68 g of3-(8,9-dihydroxynonyl)-1-methylthymine (91% yield).

A mixture of 3-(8,9-dihydroxynonyl)-1-methylthymine (2.16 g, 7.6 mmol),prepared above, and a 30% solution of hydrogen bromide in acetic acid(4.5 ml, 23 mmol) was stirred for 1 hour. The reaction mixture was addedslowly to a beaker containing sodium bicarbonate (8.4 g, 0.1 mol), icewater (30 ml), and dichloromethane (30 ml). The layers were separated,and the aqueous layer extracted with two 60 ml aliquots ofdichloromethane. The combined organic layers were washed with water (30ml) and saturated aqueous salt solution (30 ml). The washed organiclayers were then dried over sodium sulfate. Evaporation of the solventproduced 2.59 g of a slightly orange oil, inventive compound no. 1908(85% yield).

EXAMPLE 5

This example is a method of synthesis for inventive compound no. 2573(see above for chemical name and compound). A mixture of theobromine(17.64 g, 98 mmol) and sodium hydride (2.35 g, 98 mmol) indimethylsulfoxide (250 ml) was stirred for 15 minutes. After addition of9-bromo-1-nonene (20.0 g, 98 mmol, available from Alfebro) stirring wascontinued at ambient temperature for 3 days. The reaction mixture wasthen poured into water (300 ml) and extracted with four 200 ml aliquotsof dichloromethane. The combined organic layers were washed with two 150ml aliquots of saturated aqueous salt solution and the washed layersdried over sodium sulfate. Evaporating the solvent under vacuum resultedin a thick oil, which resulted in 24.34 g of white crystals aftercooling a solution of the thick oil in a minimum of dichloromethane andether 1-(8-nonenyl)-3,7-dimethylxanthine (77.5 mmol, 99% yield).

A solution of 1-(8-nonenyl)-3,7-dimethylxanthine (810 mg, 2.7 mmol),prepared above, 4-methylmorpholine-N-oxide (340 mg, 2.9 mmol) and 3drops of 2.5% osmium tetroxide in t-butanol, acetone (20 ml) and water(20 ml) was stirred for 24 hours, followed by addition of saturatedaqueous sodium dithionite solution (5 ml). After stirring the resultingmixture for 15 minutes, the reaction mixture was extracted with four 50ml aliquots of 25% ethanol-dichloromethane. The combined organic layerswere dried over sodium sulfate, and the solvent evaporated under vacuum.A resulting solid residue was recrystallized in ethanol-chloroform,producing 490 mg of 1-(8,9-dihydroxynonyl)-3,7-dimethylxanthine (54%yield).

A mixture of 1-(8,9-dihydroxynonyl)-3,7-dimethylxanthine, preparedabove, and 30% hydrogen bromide in acetic acid (0.8 ml, 3.90 mmol) wasstirred for 90 minutes. The solution was poured into a mixture of water(10 ml), sodium bicarbonate (1.35 g, and dichloromethane (10 ml). After10 minutes of vigorous stirring, the layers were separated and theaqueous portion was extracted with three 15 ml aliquots ofdichloromethane. The combined organic phases were dried over sodiumsulfate and the solvent was evaporated under vacuum, leaving 550 mg of ayellow oil, 1-(8-acetoxy-9-bromononyl)-3,7-dimethylxanthine (96% yield).Without further purification, the oil was dissolved in methanol (5 ml),to which a 1 M solution of sodium methoxide in methanol (4.1 ml, 4.1mmol) was added. After 30 minutes, the reaction mixture was poured intowater (30 ml) was extracted with three 40 ml aliquots ofdichloromethane. The combined organic layers were dried over sodiumsulfate. Evaporating the solvents under vacuum left a solid residue.Recrystallization in dichloromethane-petroleum ether yielded 380 mg of1-(8,9-oxidononyl)-3,7-dimethylxanthine (91% yield).

A mixture of 1-(8,9-oxidononyl)-3,7-dimethylxanthine (0.50 g, 1.6 mmol),prepared above and lithium perchlorate (166 mg, 1.6 mmol) was stirred inanhydrous acetonitrile (40 ml). After addition of dodecylamine (1.48 g,8.0 mmol, available from Aldrich), the mixture was stirred at reflux for4 hours. After cooling, dichloromethane (50 ml) was added and themixture was washed with water (30 ml) and saturated aqueous saltsolution (30 ml), and then dried over sodium sulfate. The solvent wasremoved under vacuum, leaving a white residue. Further purification bychromatography over silica using a dichloromethane/5%methanol eluant,produced 263 mg of a white solid, inventive compound no. 2573 (33%yield).

EXAMPLE 6

This example is a method of synthesis for inventive compound no. 3508.Triphenylphosphine (5.24 g, 20 mmol) was added incrementally to asolution of oleyl alcohol (5.37 g, 20 mmol) and carbontetrabromide (6.63g, 20 mmol) in 400 ml of dichloromethane, the resulting reaction mixturebeing stirred for an hour at room temperature. Removing the solventunder reduced pressure, left a residue, which was extracted with three200 ml aliquots of hexane. Further purification by flash chromatographyover silica gel using a hexane eluant produced 5.82 g of1-bromo-9-octadecene (88% yield).

Sodium hydride (95%, 84 mg, 3.5 mmol) was added to a solution oftheobromine (0.595 g, 3.2 mmol) in dimethylsulfoxide (15 ml). After 20minutes of stirring, 1-bromo-9-octadecene (0.995 g, 3 mmol), preparedabove, was added. After 6 hours of stirring at room temperature, thereaction mixture was warmed to 60° C. over 3 hours and then poured intoa separatory funnel containing 50 ml of water. The reaction mixture wasextracted with five 40 ml aliquots of dichloromethane. The organicextracts were combined, washed with water (50 ml) and brine (50 ml) anddried over anhydrous magnesium sulfate. Removing the solvent underreduced pressure resulted in a crude product further purified by flashchromatography over silica gel using a 30% acetone/petroleum ethereluant, yielding 0.44 g of 1-(9-octadecenyl)-3,7-dimethylxanthine (34%yield).

A solution of 1-(9-octadecenyl)-3,7-dimethylxanthine (0.15 g, 0.35mmol), 4-methylmorpholine-N-oxide (49 mg, 0.42 mmol, 1.2 equivalents.)and potassium osmate dihydrate (1 mg) in acetone (4 ml) and water (1 ml)was stirred for 6 hours. A solution of 20% aqueous sodium sulphite (2ml) was added and stirred for 30 minutes. The reaction mixture wasextracted with four 10 ml aliquots of 25% ethanol/dichloromethane. Thecombined organic extracts were dried over anhydrous magnesium sulfate,the solvent evaporated under reduced pressure and a residue purified byflash chromatography over silica gel using amethanol(5%)/dichloromethane eluant, yielding 0.65 g of1-(9,10-dihydroxyoctadecyl)-3,7-dimethylxanthine (40.4% yield).

A 50 ml RB flask fitted with a dropping funnel, magnetic stirring barand an argon inlet was placed in a solution of1-(9,10-dihydroxyoctadecyl)-3,7-dimethylxanthine (464 mg; 1 mmol) andtriphosgene (148.37 mg; 0.5 mmol) in anhydrous dichloromethane. Theresulting mixture was cooled to 0° C. A solution of pyridine (58.2 mg; 2mmol) in anhydrous dichloromethane (3 ml) was added dropwise and thereaction mixture was warmed to room temperature and stirred for 6 hours.The reaction mixture was then diluted with water (20 ml) and extractedwith three 50 ml aliquots of dichloromethane. The combined organicextract was washed with water (50 ml), saturated copper sulphatesolution (50 ml), water (50 ml), and brine solution (50 ml) and driedover anhydrous magnesium sulfate. Evaporating the solvent under reducedpressure left a residue which was further purified by flashchromatography over silica gel using a 50% ethyl acetate/hexane eluant,resulting in 200 mg of compound no. 3508 (40.8% yield).

EXAMPLE 7

This example is a method of synthesis for inventive compound no. 3537.Sodium hydride (95%, 1.26 g, 50 mmol) was added to a solution oftheobromine (7.2 g, 40 mmol) in dimethylsulfoxide (300 ml). After 20minutes of stirring, undecenylmesylate (7.95 g, 30 mmol) was added andthe resulting mixture stirred for 12 hours at room temperature. Thereaction was warmed to 70-80° C. and stirred for 4 hours. The reactionmixture was then poured into a separatory funnel containing water (1 L)and extracted with five 200 ml aliquots of dichloromethane. The organicextracts were combined, washed with water (100 ml) and brine (100 ml)and dried over anhydrous magnesium sulfate. The solvent was evaporatedunder reduced pressure, resulting in a crude product, which was furtherpurified by flash chromatography over silica gel using a 20%hexane/dichloromethane eluant producing 4.6 g of1-(10-undecenyl)-3,7-dimethylxanthine (46.3% yield).

A solution of 1-(10-undecenyl)-3,7-dimethylxanthine (4.3 g, 13 mmol),prepared above, 4-methylmorpholine-N-oxide (1.942 g, 16.6 mmol) andpotassium osmate dihydrate (9.5 mg, 0.026 mmol) in acetone (45 ml) andwater (10 ml) was stirred for 6 hours. A solution of 20% aqueous sodiumsulphite (12 ml) was added and stirred for 30 minutes. The reactionmixture was extracted with four 100 ml aliquots of 25%ethanol/dichloromethane. The combined organic extracts were dried overanhydrous magnesium sulfate. Evaporating the solvent under reducedpressure left a residue, which upon subsequent purification by flashchromatography over silica gel using a methanol (5%)/dichloromethaneeluant produced 3.6 g of1-(10,11-dihydroxyundecanyl)-3,7-dimethylxanthine (76% yield).

1-(10,11-Dihydroxyundecanyl)-3,7-dimethylxanthine (3.6 g, 10 mmol) wasstirred with hydrogen bromide (6.2 ml, 8.4 g of a 30% solution in aceticacid, 31.1 mmol) for 90 minutes. The mixture was then added to a flaskcontaining 100 ml aqueous sodium bicarbonate solution and 75 mldichloromethane. After 10 minutes of vigorous stirring, the layers wereseparated and the aqueous portion washed with three 75 ml aliquots ofdichloromethane. The organic portions were combined and dried overmagnesium sulfate. Evaporating the solvent left 3.6 g of1-(10-acetoxy-11-bromoundecanyl)-3,7-dimethylxanthine. Without furtherpurification, 1-(10-acetoxy-11-bromoundecanyl)-3,7-dimethylxanthine wastaken up in 25 ml of methanol and treated with a solution of sodiummethoxide (prepared from 0.28 g, 12.2 mmol sodium, and 25 ml methanol).After 30 minutes, most of the solvent was removed under reduced pressureand the residue was extracted with three 75 ml aliquots ofdichloromethane. The organic portions were combined and dried overmagnesium sulfate. The solvent was evaporated under reduced pressure,leaving an off-white solid. Further purification of the off-white solidby column chromatography over silica gel using a dichloromethane/(3%)methanol eluant provided 2.0 g of1-(10,11-oxidoundecanyl)-3,7-dimethylxanthine (57.5% yield).

Octylamine (3.4 ml, 21 mmol) was added to a stirring mixture of1-(10,11-oxidoundecyl)-3,7-dimethylxanthine (5.00 g, 14.4 mmol),prepared above, and lithium perchlorate (1.69 g, 16 mmol) in anhydrousacetonitrile (60 ml). Stirring was continued for 16 hours at 50° C.After cooling to ambient temperature, water (100 ml) was added, and themixture was extracted with three 100 ml aliquots of dichloromethane-10%methanol. The combined organic extracts were washed with aqueoussaturated salt solution (150 ml) and dried over sodium sulfate. Thesolvent was removed under vacuum, leaving a solid, which wasrecrystallized twice in dichloromethane/ether/hexane, producing 5.76 gof a white powder,1-(11-octylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (84% yield).

A solution of 1-(11-octylamino-10-hydroxyundecyl)-3,7-dimethylxanthine(1.70 g, 3.0 mmol), prepared above, in acetic anhydride (5 ml) washeated for 2 hours at 90° C. After cooling, methanol (10 ml) was addedand the mixture was stirred for 30 minutes. After addition of water (20ml), the mixture was extracted with three 40 ml aliquots ofdichloromethane. The combined organic layers were washed with water (15ml) and saturated aqueous salt solution (15 ml). After the solution wasdried over sodium sulfate, the solvent was evaporated, leaving a yellowoil residue. The residue was further purified by chromatography overneutral activity II alumina using a dichloromethane-5% methanol eluant,resulting in 1.43 g of a colorless oil, inventive compound no. 3537 (2%yield), which solidified upon standing.

EXAMPLE 8

This example is a method of synthesis for inventive compound no. 3541(see above for chemical name and structure). Sodium hydride (312 mg, 13mmol) was added to a solution of octanol (10 ml) in toluene (20 ml).After bubbling ceased, 1-(10,11-oxidoundecanyl)-3,7-dimethylxanthine(2.50 g, 7.2 mmol), prepared in example 7 above, was added to themixture, which was subsequently stirred for 3 hours at 60-70° C. Aftercooling, the mixture was added to a solution of saturated aqueoussolution of ammonium chloride (15 ml) and water (10 ml) and extractedwith three 50 ml aliquots of dichloromethane. The combined organiclayers were washed with saturated aqueous salt solution and dried oversodium sulfate. Evaporation of the solvents under vacuum left a solidresidue, which when purified by chromatography over neutral activity IIalumina using a dichloromethane eluant produced recovered epoxide (411mg) and 1.34 g of 1-(11-octyloxy-10-hydroxyundecyl)-3,7-dimethylxanthine(49% yield).

A mixture of 1-(11-octyloxy-10-hydroxyundecyl)-3,7-dimethylxanthine(0.31 g, 0.6 mmol), prepared above, and acetic anhydride (4 ml) washeated at 90° C. for 2 hours. After cooling to ambient temperature,dichloromethane (40 ml) and saturated sodium bicarbonate solution (50ml) were added. The organic layer was separated, and the aqueous layerwas extracted with dichloromethane (50 ml). The combined organic layerswere washed with water (10 ml) and saturated aqueous salt solution (10ml). After the solution was dried over sodium sulfate, the solvent wasremoved, leaving an oily residue. The residue was purified bychromatography over silica using a dichloromethane-10% methanol eluant,producing 149 mg of inventive compound no. 3541 (49% yield).

EXAMPLE 9

This example is a method of synthesis for inventive compound no. 3549(see above for chemical name and structure). A solution of11-bromoundecanoic acid (5.70 g, 22 mmol, available from Aldrich) andp-toluenesulfonic acid (0.1 g) in absolute ethanol (100 ml) was refluxedfor 3 hours. A saturated aqueous sodium bicarbonate solution (40 ml) wasadded and the reaction mixture then extracted with three 70 ml aliquotsof dichloromethane. The combined extracts were washed with water (50 ml)and saturated aqueous salt solution (50 ml) and the solvent wasevaporated, leaving a colorless oil. Ethyl 11-bromoundecanoate (5.92 g,94% yield) was collected during distillation (2 mm) at 135° C. Asolution of this bromoester (5.92 g, 20 mmol) and 1-sodiotheobromine(4.08 g, 20 mmol) in dimethylsulfoxide (80 ml) was stirred for 18 hoursat ambient temperature. The mixture was added to water (100 ml) anddichloromethane (100 ml). The aqueous layer was extracted with two 80 mlaliquots of dichloromethane. The combined organic layers were washedwith water (80 ml) and saturated aqueous salt solution (80 ml), driedover magnesium sulfate, and the solvent was evaporated under vacuum,leaving a white solid residue. The residue was recrystallized indichloromethane/ether/hexane, yielding 4.95 g of 1-(ethyl11-yl-undecanoate)-3,7-dimethylxanthine (62% yield).

A solution of potassium hydroxide (0.50 g, 9.0 mmol) in water (1 ml) wasadded to a stirring suspension of 1-(ethyl11-yl-undecanoate)-3,7-dimethylxanthine (2.52 g, 6.4 mmol), preparedabove, in methanol (15 ml). The mixture was warmed until homogeneous,and the stirring was continued overnight at ambient temperature. Water(10 ml) was added to the reaction mixture, followed by a 5% solution ofsulfuric acid (10 ml). The precipitate was filtered off and washed withether, then dried under vacuum, resulting in 2.12 g of inventivecompound no. 3549 (91% yield).

EXAMPLE 10

This example is a method of synthesis for inventive compound no. 3554(see above for chemical name and structure). A solution of1-(11-yl-undecanoic acid)-3,7-dimethylxanthine (1.62 g, 4.5 mmol),prepared in example 10 above, and thionyl chloride (0.5 ml, 6.7 mmol) intoluene (5 ml) was heated at 80° C. for 1 hour and then cooled. Thesolvent was evaporated under a nitrogen stream. The resulting acidchloride was taken up in dichloromethane (20 ml), and 1-octylamine (2ml, 11 mmol) was added by syringe to the stirring solution. After 2hours, water (50 ml) was added and the mixture was extracted with three50 ml aliquots of dichloromethane. The combined organic extracts werewashed with 5% hydrochloric acid (100 ml) and saturated aqueous saltsolution (60 ml) and then dried over sodium sulfate. The solvent wasevaporated under vacuum, leaving a residue, which was further purifiedby chromatography over basic activity II alumina using adichloromethane/10% methanol eluant, yielding 1.47 g of compound no.3554 as a white solid (69% yield).

EXAMPLE 11

This example is a method of synthesis for inventive compound no. 3564(see above for chemical name and structure). Tetradecylamine (797 mg,3.7 mmol) was added to a stirring mixture of1-(10,11-oxidoundecanyl)-3,7-dimethylxanthine (1.00 g, 2.9 mmol),prepared in example 7 above, and lithium perchlorate (309 mg, 2.9 mmol)in anhydrous acetonitrile (20 ml). Stirring was continued for 4 hours at60° C. After cooling to ambient temperature, water (50 ml) was added,and the mixture was extracted with three 100 ml aliquots ofdichloromethane. The combined organic extracts were washed with aqueoussaturated salt solution and dried over sodium sulfate. The solvent wasremoved under vacuum, leaving a solid residue, which was purified bychromatography over neutral activity II alumina using adichloromethane-3% methanol eluant, resulting in 550 mg of a whitepowder, 1-(11-tetradecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine(34% yield).

A solution of1-(11-tetradecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (600 mg,1.1 mmol) and acetic anhydride (0.6 ml, 6.4 mmol) in pyridine (15 ml)was stirred at ambient temperature for 20 hours. After addition ofdichloromethane (100 ml) the mixture was washed with two 50 ml aliquotsof 10% aqueous hydrochloric acid and saturated aqueous salt solution (50ml), and then dried over magnesium sulfate. The solvent was removedunder vacuum, leaving a residue, which was then purified bychromatography over neutral activity II alumina using adichloromethane-3% methanol eluant, resulting in 475 mg of inventivecompound no. 3564 (69% yield).

EXAMPLE 12

This example is a method of synthesis for inventive compound no. 3577(see above for chemical name and structure). Under an argon atmosphere,oxalyl chloride (0.72 ml, 8.3 mmol) was added to a slurry of1-(11-yl-undecanoic acid)-3,7-dimethylxanthine (2.0 g, 5.5 mmol),prepared in example 9 above, in dichloromethane (20 ml). The reactionwas heated to reflux and allowed to stir for 1 hour. The resultingsolution was cooled to ambient temperature and then slowly transferredto a stirring solution of 3,4,5-trimethoxybenzylamine (2.8 ml, 16.5mmol) in dichloromethane (20 ml), followed by cooling to 0° C. After 2hours of stirring at ambient temperature, the reaction was poured into3% aqueous hydrogen chloride solution (100 ml), followed by saturatedaqueous salt solution (40 ml). The mixture was extracted with three 50ml aliquots of dichloromethane. The combined organic layers were washedwith saturated aqueous salt solution (50 ml) and dried over magnesiumsulfate. The solvents were evaporated under reduced pressure, leaving acrude yellow residue. Column chromatography over alumina using an ethylacetate/ ethyl acetate-methanol eluant and subsequent recrystallizationfrom ethyl acetate produced 0.98 g of a white solid, inventive compoundno. 3577 (33% yield).

EXAMPLE 13

This example shows an inhibitive effect of inventive compounds nos. 3549and 3546 on murine thymocyte proliferation stimulated by Concanavalin A(ConA) and interleukin-2 (IL-2). This assay is an in vitro, predictivemodel of a compound's therapeutic potential in treating or preventingautoimmune, immune or inflammatory diseases. Procedurally, thymuses wereobtained from normal, female Balb/C mice. The thymuses were dissociatedand plated into 96-well plates at a density of 2×10⁵ cells/well. ConA(0.25 mg/ml) and IL-2 (12.5 ng/ml) were added to the wells. Drug wasadded at various doses two hours prior to activation with ConA and IL-2.The cells were incubated for 4 days at 37° C. On day 4, the cells werepulsed with tritiated thymidine and allowed to incubate for anadditional 4 hours. Harvested cells were analyzed for incorporatedtritiated thymidine, determined using a liquid scintillation counter.Dose response curves were prepared from the assay results and used tocalculate an IC50 value for each compound tested.

In representative dose response curves prepared for assays investigatingcompounds nos. 3546 and 3549, FIGS. 1 and 2, respectively, illustratethe inhibitive effects of these compounds on proliferation of thymocytesstimulated with ConA and IL-2. Background counts, without addition ofrepresentative inventive compounds were about 190 cpm. FIG. 1illustrates a remarkable ability of inventive compound no. 3546 toinhibit proliferation of thymocytes in this system. FIG. 2 illustrates aless pronounced ability of the inventive compounds to inhibit thymocyteproliferation, suggesting specificity of particular inventive compoundsfor treating specific diseases. As shown, inventive compound no. 3546inhibited ConA/IL-2 stimulated proliferation at compound concentrationsless than 20 μM, with an IC50 value, experimentally calculated from thisdose response curve, of about 4.8 μM. These concentrations plotted arewithin concentrations known to be achieved in vitro for treatingdisease.

EXAMPLE 14

This example illustrates an ability of inventive compounds nos. 1514 and1583 to inhibit proliferation of peripheral blood mononuclear cells(PBMC) in response to allogeneic stimulation. This in vitro mixedlymphocyte reaction (MLR) assay is useful in assessing biologicalactivity of an inventive compound. Procedurally, PBMC were obtained bydrawing whole blood from healthy volunteers in a heparinized container,the whole blood samples diluted with an equal volume of hanks balancedsalt solution (HBSS).

This mixture was layered on a sucrose density gradient, such as aFicoll-Hypaque® gradient (specific gravity 1.08), and centrifuged(1000×g) for 25 minutes at no warmer than room temperature. PBMC wereobtained from a band at a plasma-Ficoll interface, separated and washedat least twice in a saline solution, such as HBSS. Contaminating redcells are lysed, for example, by ACK lysis for 10 minutes at 37° C., andthe PBMC were washed twice in HBSS. The pellet of purified PBMC wasresuspended in complete medium, such as RPMI 1640 plus 20% humaninactivated serum.

Proliferative response of PBMC to allogeneic stimulation was determinedin a two-way MLR performed in a 96-well microtiter plate. Approximately10⁵ test-purified PBMC in 200 μl complete medium were co-cultured withapproximately 10⁵ autologous (control culture) or allogeneic (stimulatedculture) PBMC. Allogeneic cells were from HLA disparate individuals.Varying doses of compounds nos. 1514 and 1583 were added simultaneouslyupon addition of cells to the microtiter plate. The cultures wereincubated for 6 days at 37° C. in a 5% CO₂ atmosphere, after which time,tritiated thymidine was added (for example, 1 μCi/well of 40 to 60Ci/mmole) and proliferative inhibition was assessed by determiningamount of tritiated thymidine taken up, using liquid scintillationcounting.

FIGS. 3 and 4 are plotted graphs of compound concentrations (μM) versusinhibition (as a function of incorporated thymidine, cpm) for compoundsnos. 1514 and 1583, respectively. FIGS. 3 and 4 illustrate an ability ofthe inventive compounds tested to inhibit PBMC proliferation. Atconcentrations less than 250 μM, compound no. 1583 more significantlyinhibited incorporation of thymidine. Similarly, although to lesserdegrees in comparison to compound no. 1583, compound no. 1514 inhibitedproliferation in this MLR assay at compound concentrations less than 250μM.

EXAMPLE 15

This example illustrates inhibitive effects of the inventive compoundson Balb/3T3 cell proliferation in response to platelet derived growthfactor (PDGF) stimulation.

Disregulated PDGF-proliferative response has been linked to a variety ofdiseases, including, e.g., restenosis, atherosclerosis, fibrosis, andtumor cell angiogenesis. Balb/3T3 cells respond vigorously to PDGFstimulation, and are useful in vitro models for further study ofPDGF-induced proliferation. In an assay useful in determining whether acompound would be useful in treating diseases characterized by this orsimilar disregulated proliferative responses, research indicates thatmany of the inventive compounds inhibit PDGF-induced proliferation ofBalb/3T3 cells.

Balb/3T3 cells were plated in low serum-containing medium for 24 hoursprior to stimulation with various concentrations of inventive compound.Specifically, in this assay, inventive compounds nos. 1529, 2538, 3537,3542, 3546, 3554, 3557, 3559, 3562, 3564, 3571, 3573 and 3577 weretested. PDGF was added at varying concentrations along with tritiatedthymidine. The cells were allowed to incubate for one day, followingaddition of PDGF and thymidine. 24 hours later, the cells were harvestedand counted by liquid scintillation counting. Data obtained for eachcompound were plotted as % inhibition versus concentration of inventivecompound and IC50 values experimentally calculated from the resultsplotted.

In conjunction with the Balb/3t3 proliferation assay, a relatedviability assay was conducted to assess the cytotoxicity of compoundswhich inhibit proliferation in this system. The assay protocol wasidentical to that performed above except that tritiated thymidine wasnot added after the 24 hour incubation with PDGF. Subsequent toincubation, a 10 μM solution of2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein, acetoxymethyl ester(BCECF--a compound that when cleaved by esterases, yields a fluorescentproduct, thus providing a measure of cell number) was added and thecells incubated for 30 minutes at 37° C. Following this incubation,BCECF was replaced with PBS and the plate read for fluorescence in aMillipore "cytofluor". Data obtained were plotted as a percent ofcontrol versus concentration of inventive compound tested and fiftypercent (50%) lethal dose concentrations (LD50) for the inventivecompounds tested were experimentally calculated from the plotted data.

FIG. 5 reports the experimentally calculated IC50 values obtained in theforegoing proliferation assay and LD50 values obtained in thecorresponding viability assay for each inventive compound tested. Thereported results indicate that many of the inventive compounds have IC50values--the concentration of inventive compound in the proliferationassay inhibiting 50% proliferation of a control level--less than 10 μM.Specifically, inventive compounds nos. 3554, 3559, 3571 and 3577 haveIC50 values at or below 1 μM. Of significance, compound no. 3577inhibits 50% proliferation at an extremely low concentration of 0.1 μM!

LD50 values reported in viability assays for the inventive compoundstested indicate that many of the compounds have LD50 values abovemeasurable levels. In FIG. 5, experimentally calculated IC50 valueswhich equaled or exceeded 20 μM were reported as 20 μM. For a majorityof compounds tested, a significant concentration interval exists betweenthe IC50 and LD50 experimentally calculated, indicating that theinventive compounds are not only candidates for treating or preventingrestenosis, atherosclerosis, fibrosis, tumor cell angiogenesis and othersimilar diseases, but possess significant windows for therapeutictreatment.

What is claimed is:
 1. A therapeutic compound, including resolvedenantiomers, diastereomers, salts, or solvates thereof, having theformula:

    CORE MOIETY --(R).sub.j

wherein: j is an integer from one to three; the core moiety isxanthinyl; and R is a member selected from the group consisting ofhydrogen, hydroxyl, amino, C.sub.(1-10) alkyl, C.sub.(2-10) alkenyl,carbocyclic group and heterocyclic group and at least one R havingformula I: ##STR8## with the proviso that the R having formula I is notattached at the N₉ nitrogen atom of the xanthinyl;wherein: p is two; nis an integer from seven to twenty; (CH_(p))_(n) is unsubstituted orsubstituted by a member selected from the group consisting of halogen,hydroxyl, C.sub.(1-10) allyl, C.sub.(2-10) alkenyl, C.sub.(1-10)alkoxyl, C.sub.(1-10) alkanoyloxyl, C.sub.(1-10) oxoalkyl, carbocyclicgroup and heterocyclic group; R₁ is a member selected from the groupconsisting of CH₂ ; NR₃, R₃ being hydrogen, C.sub.(1-20) alkyl,C.sub.(1-20) alkoxyl, C.sub.(2-20) alkenyl, C.sub.(1-20) hydroxyalkyl,or carbocyclic or heterocyclic group; O; --CHR₄ O-- or --C(R₄)_(r) O--,r being one or two, R₄ being ═O, hydrogen, C.sub.(1-20) alkyl,C.sub.(1-20) alkoxyl, C.sub.(2-20) alkenyl, C.sub.(1-20) hydroxyalkyl,C.sub.(1-20) aminoalkyl, --(CH₂)_(q) A(R₅)_(m), q being an integer fromone to four, A being N or O, m being one or two and R₅ being hydrogen,C.sub.(1-10) alkyl, C.sub.(1-10) alkoxyl, C.sub.(2-10) alkenyl,C.sub.(1-10) hydroxyalkyl, C.sub.(1-10) aminoalkyl, carbocyclic group orheterocyclic group; R₂ is a member selected from the group consisting ofhydrogen, halogen, C.sub.(1-10) alkyl, C.sub.(1-10) alkoxyl,C.sub.(2-10) alkenyl, C.sub.(1-10) hydroxyalkyl, C.sub.(1-20)aminoalkyl, --A(R₅)_(m) and --CHR₆ A(R₅)_(m) ; wherein A, R₅ and m areas defined above, R₆ is a C.sub.(1-20) alkyl, C.sub.(1-20) alkoxyl,C.sub.(2-20) alkenyl, C.sub.(1-20) hydroxyalkyl, C.sub.(1-20)aminoalkyl, carbocyclic group or heterocyclic group, or A is N, m is twoand the two R₅ join together with the A to form a heterocycle havingfrom four to seven ring atoms selected from the group consisting of N, Cand O, the A comprising a heteroatom of the heterocycle; wherein atleast one of: 1) R₁ is NR₃, O, or --CHR₄ O-- or 2) R₂ is --A(R₅)_(m) ;and wherein said carbocyclic group or heterocyclic group is a memberselected from the group consisting of: anthracenyl,bicyclo[4.4.0]decanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.0]heptanyl,bicyclo[4.1.0]heptanyl, bicylo[2.2.1]hexanyl, bicyclo[4.3.0]nonanyl,bicyclo[2.2.2]octanyl, biphenyl, cyclopentadienyl, cyclopentanyl,cyclobutanyl, cyclobutenyl, cycloheptanyl, cyclohexanyl, cyclooctanyl,cyclopropanyl, fluorenyl, indenyl, phenyl, quinonyl, napthalenyl,phenanthrenyl, azetidinyl, benzofuranyl, benzothiophenyl, carbazolyl,furanyl, glutarimidyl, indolyl, isoquinolinyl, oxazolyl, oxetanyl,oxiranyl, pyrrolidinyl, pyranyl, piperidinyl, pyridinyl, pyrrolyl,quinolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyland thiophenyl.
 2. A compound selected from the group consisting of:##STR9##
 3. A pharmaceutical composition comprising a compound accordingto claim 1 and a suitable carrier, diluent or excipient.
 4. The compoundaccording to claim 1, wherein n is an integer from seven to sixteen. 5.The pharmaceutical composition of claim 3, wherein the composition isformulated for parenteral, topical or oral administration or forinhalation.
 6. The compound according to claim 1, wherein j is one. 7.The compound according to claim 1, wherein R having formula I isattached to at least one of N₁ or N₃ xanthinyl nitrogens, the N₇xanthinyl nitrogen being substituted by a member selected from the groupconsisting of hydrogen, methyl or amino.
 8. The compound according toclaim 1, wherein R having formula I is bonded to an N₁ nitrogen ofxanthinyl, and wherein an N₃ and an N₇ xanthinyl nitrogens areindependently substituted by a member selected from the group consistingof hydrogen, C.sub.(1-6) alkyl and amino.
 9. A compound of the followingformula:
 10. A compound of the following formula:
 11. A compound of thefollowing formula:
 12. A compound of the following formula:
 13. Acompound of the following formula:
 14. A compound of the followingformula: